Immune cells derived from induced pluripotent stem cell

ABSTRACT

Embodiments disclosed here are production methods and compositions of engineered immune cells, such as B or T lymphocytes, from limited lineage myeloid progenitor cells, or from pluripotent stem cells, or from multilineage hematopoietic progenitor cells comprising the addition of various cell differentiation transcription factors and inhibiting epigenetic histone methylations in said cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional under 35 U.S.C. § 121 of co-pendingU.S. application Ser. No. 16/330,307 filed Mar. 4, 2019, which is a 35U.S.C. § 371 National Phase Entry Application of InternationalApplication No. PCT/US2017/050167 filed Sep. 6, 2017, which designatedthe U.S., and which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 62/383,984 filed on Sep. 6, 2016, thecontents of each of which are incorporated herein by reference in theirentireties.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersHL100001 and DK092760 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Oct. 26, 2022, isnamed 701039-087011USD1_SL.xml and is 146,613 bytes in size.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to the fields of medicine, cellbiology, and molecular biology. This disclosure relates to productionmethods of immune cells such as B or T lymphocytes from limited lineagemyeloid progenitor cells, or from pluripotent stem cells (PSCs), or frommultilineage hematopoietic progenitor cells (MHPCs).

BACKGROUND

There is a lack of supply of functional immune cells for the in vivocellular replacement therapy, therapy for a host of diseases, disordersand conditions, and for the in vitro studies of disease modeling, drugscreening, and hematological diseases. Bone marrow transplantation is byfar the most established cellular replacement therapy for a variety ofhematological disorders. The functional unit of a bone marrow transplantis the hematopoietic stem cell (HSC), which resides at the apex of acomplex cellular hierarchy and replenishes blood development throughoutlife. However, the scarcity of HLA-matched HSCs or patient-specific HSCsseverely limits the ability to carry out transplantation, diseasemodeling, drug screening, and in vitro studies of hematologicaldiseases. Often, there is not a large enough cell populationtransplanted into a recipient subject to ensure sufficient engraftmentand reconstitution in vivo in the recipient subject.

As such, many studies have been developed to generate HSCs fromalternative sources. For example, reprogramming of somatic cells toinduced pluripotent stem cells (iPSCs) has provided access to a widearray of patient-specific pluripotent cells, a promising source fordisease modeling, drug screens and cellular therapies. Pluripotent cellsare induced in human and mouse somatic cells by the forced expression ofOCT4 (Oct4) and SOX2 (Sox2) with either the combinations of KLF4 (Klf4)and optionally c-MYC (c-Myc), or the combinations of NANOG (Nanog) andLIN28 (Lin28). Alternative combinations of transactiving factors includeOCT4, SOX2, NANOG and LIN28. Mouse iPS cell lines derived from bonemarrow hematopoietic progenitor cells (HPCs) has been reported.Derivation of human iPS cells from postnatal human blood cells, fromgranulocyte colony-stimulating factor (G-CSF) mobilized peripheral bloodCD34+ cells, and from human cord blood and adult bone marrow CD34+ cellswithout any pre-treatment such as G-CSF mobilization has been alsoreported. These reports all employed HPCs, stem cells as the source ofiPSCs. Somatic cells such as T lymphocyte cells, B lymphocyte cells,fibroblasts and keratinocytes are also used as the alternative sourcesof iPSCs.

The iPSCs have been shown to differentiate into various cells belongingto the three germ layers, as demonstrated by the analysis of teratomasgenerated from human iPSCs and mouse iPSCs. In addition, thepluripotency of iPSCs is confirmed by the contribution of iPScell-derived cells to various organs of the chimeric mice developed fromiPSC-introduced blastocysts.

However, in addition to the cell quantity and cell source problems,there is still a big hurdle in producing iPSCs-derived hematopoieticstem and progenitor cells (iPSCs-HSPC) or the differentiated cellstherefrom where the progeny cells would engraft in vivo. As describedabove, the various studies aimed at in vitro generating HSCs fromalternative sources produced hematopoietic progenitor or stem cells thatdo not engraft well in vivo.

SUMMARY

Embodiments of the present disclosure relate to methods for producingpatient-specific, histocompatible, multipotent hematopoietic progenitorcells (MHPCs) that can be subjected to specific, directeddifferentiation to provide functional immune cells in quantities largerthan what has been traditionally possible in in vitro cultureconditions. Embodiments of the present disclosure also relatecompositions comprising these MHPCs, and progeny cells resulting fromthe specific, directed differentiation process, and the uses of thesecells.

There is a lack of supply of functional HLA-matched immune cells for thein vivo cellular replacement therapy, the treatment of diseases,disorders and medical conditions, and for the in vitro studies ofdisease modeling, drug screening, and hematological diseases. Mostly,the immune cells are differentiated from hematopoietic stem cell (HSC)but there is a scarcity of HLA-matched HSCs. The present method solvesthis problem by reversing the lineage potentials of previouslynon-lymphoid lineage committed myeloid progenitor cells back to MI-IPCs,and then subsequently specifically promoting and directingdifferentiation of the hematopoietic progenitor cells (HPCs) into thelymphoid lineage. In addition, the MI-IPCs, having reversed lineagepotentials, are modified to have enhanced in vivo engraftment andreconstitution properties. The production method is useful, for example,as a cell preparation method in immunotherapy.

ABBREVIATIONS USED HEREIN

-   -   HPCs=hematopoietic progenitor cells    -   MHPCs=multilineage hematopoietic progenitor cells or multipotent        hematopoietic progenitor cells    -   iPSCs=induced pluripotent stem cells    -   HSCs=hematopoietic stem cells

The inventors, by introducing at least three exogenous transcriptionfactors, ERG, HOXA9, and RORA, into non-lymphoid lineage committedmyeloid progenitor cells, were able to reversed the lineage potential ofthese cells. The resultant cells were MHPCs.

The blood cells produced during hematopoiesis are divided into thefollowing three cell lineages: (1) erythroid cells, (2) lymphoid cells,and (3) myeloid cells. Erythroid cells, including normoblasts,erythroblasts and mature red blood cells (RBCs), are the most commontype of blood cell and are a principal means of delivering oxygen fromthe lungs to body tissues. Lymphoid cells, including B-cells andT-cells, are a type of white blood cell that play a significant role inthe body's immune defenses. Myeloid cells, including granulocytes,megakaryocytes, and macrophages, are a diverse group of cells comprisingother white blood cells (e.g., neutrophils, eosinophils and basophils)and platelets.

Myeloid progenitor cells are committed to the myeloid linage, which is anon-lymphoid lineage. Myeloid progenitor cells in the myeloid lineageundergo further cell division, differentiation and maturation, and themyeloid lineage produces the following cell types: megakaryocytes,thrombocytes, erythrocytes, mast cells, myeloblast, basophils,neutrophils, eosinophils, monocytes and macrophages. The myeloid lineageis different from the lymphoid lineage, which produces immune cells suchas T and B lymphocytes. By further inhibiting a histonemethyltransferase EZH1 in these reversed lineage MHPCs, the inventorswere able to direct the differentiation of these cells into immune cellsby co-culture with OP9-DL1/4 cells or by activating the Notch signalingpathway in these cells. Moreover, the inventors found that byincorporating two additional exogenous transcription factors, DACH1 andNFIA, into these cells enhanced the lymphoid potential of these cellsupon co-culture with OP9-DL1/4 cells or by activating the Notchsignaling pathway. Furthermore, the inventors found that byincorporating two other exogenous transcription factors, SOX4 and MYB,into these cells enhanced the engraftment and reconstitution of thesecells in vivo in a recipient subject.

The advantage of the disclosure protocol is that the method now enablessemi-permanent bulk production of desired and specific immune cells froma source of cells, which can be readily collected from the patient'sbody. For example, somatic cells such as blood cells, immune cells, skincells etc. The production of function immune cells is not restricted tousing only stem or progenitor cells obtained from a patient. Theproduced immune cells can then be used for immunotherapy.

Accordingly, it is an object of the present disclosure to provideproduction methods of immune cells which include the step of reversingthe lineage potentials of previously non-lymphoid lineage committedmyeloid progenitor cells to MI-IPCs, and specific and directeddifferentiating the reversed-lineage MHPCs into desired immune cells.The non-lymphoid lineage committed myeloid progenitor cells can be madefrom iPSCs, which are generated from any cells in the patient's body,e.g. somatic cells. Such cells can be readily collected from thepatient's body. For example, cells from a blood sample, a skin sample, abuccal mouth swab etc. The non-lymphoid lineage committed myeloidprogenitor cells may be harvested from the patient's bone marrow.

It is also the objective of this the present disclosure to providemethods for enhancing or improving the in vivo engraftment, orreconstitution, or both of hematopoietic related cells that have beenimplanted into a subject.

It is also the objective of this the present disclosure to providecompositions of modified (also referred to as engineered) cells for usein in vivo cellular replacement therapy, medical therapy such as cancerimmune therapy, and for the in vitro studies of disease modeling, drugscreening, and hematological diseases.

Accordingly, disclosed here is (1) a method for preparing modifiedimmune cells, such as T or B cells, the method which comprises a step ofreversing the lineage potentials of myeloid progenitor cells to HPCsusing exogenous copies of transcription factors and a step of specificand directed differentiation of the reversed lineage HPCs into immunecells; (2) modified myeloid progenitor cells having reversed lineage andhave increased lymphoid lineage potential; (3) compositions whichcontain the modified myeloid progenitor cells having reversed lineagethat include increased lymphoid lineage potential; (4) modified myeloidprogenitor cells described herein and compositions thereof for use inthe manufacture/production of described modified immune cells; (5)modified myeloid progenitor cells described herein and compositionsthereof for use in cellular replacement therapy, or for the treatment ofcancer, autoimmune disorders, hematological diseases or other geneticdiseases and disorders; (6) a pharmaceutical composition which containsthe modified immune cells that are prepared by the method describedherein; and (7) a method for treatment uses with the immune cells madewith the above-described method, such as bone marrow transplant andcancer immune therapy, autoimmune disorders, hematological diseases orother genetic diseases and disorders. The modified immune cells aremammalian cells, such as human cells.

In one embodiment, this disclosure provides a modified or an engineeredmyeloid progenitor cell having reversed lineage that include increasedlymphoid lineage potential. In one embodiment, this disclosure providesa modified or an engineered myeloid progenitor cell having reversedlineage to include increased lymphoid lineage potential that is producedby a method described herein. In some embodiment, the modified or anengineered myeloid progenitor cell has an exogenous gene coding copy ofeach of the following transcription factors: ERG, HOXA9, and RORA, viaERA transfections. In one embodiment, the modified or engineered myeloidprogenitor cell further comprises an exogenous gene coding copy of SOX4,or MYB, or both SOX4 and MYB. In another embodiment, the modified orengineered myeloid progenitor cell further comprises an exogenous genecoding copy of DACH1, or NFIA, or both DACH1 and NFIA. In someembodiment, the modified myeloid progenitor cells are derived fromlineage-restricted CD34⁺CD45⁺ myeloid precursor cells.

In another embodiment, this disclosure provides a composition comprisingmodified or engineered myeloid progenitor cell described herein.

In another embodiment, this disclosure provides modified myeloidprogenitor cells described herein and compositions thereof for use inthe manufacture/production of described modified immune cells, whereinthe modified myeloid progenitor cell comprises an exogenous gene codingcopy of each of the following transcription factors: ERG, HOXA9, andRORA. In one embodiment, the modified or engineered modified myeloidprogenitor cell further comprises an exogenous gene coding copy of SOX4,or MYB, or both SOX4 and MYB. In another embodiment, the modified orengineered modified myeloid progenitor cell further comprises anexogenous gene coding copy of DACH1, or NFIA, or both DACH1 and NFIA.

In another embodiment, this disclosure provides modified myeloidprogenitor cells described herein and compositions thereof for use incellular replacement therapy, or for the treatment of cancer, autoimmunedisorders, hematological diseases, or other genetic diseases anddisorders, wherein the modified myeloid progenitor cell comprises anexogenous gene coding copy of each of the following transcriptionfactors: ERG, HOXA9, and RORA. In one embodiment, the modified orengineered modified myeloid progenitor cell further comprises anexogenous gene coding copy of SOX4, or MYB, or both SOX4 and MYB. Inanother embodiment, the modified or engineered modified myeloidprogenitor cell further comprises an exogenous gene coding copy ofDACH1, or NFIA, or both DACH1 and NFIA.

Accordingly, in one embodiment, provided herein is a method comprising(a) in vitro or ex vivo generating multilineage hematopoietic progenitorcells (MHPCs) from myeloid progenitor cells; (b) inhibiting a histonemethyltransferase in the resultant population of MHPCs; and, (c)differentiating the resultant population of MHPCs in the presence of anotch ligand or defined stromal cells or both to promote differentiationinto the lymphoid lineage. In some embodiments, in vitro culturing ofthe cells occurs between step (a) and step (b). In some embodiments,selection of cells occurs between step (a) and step (b).

In another embodiment, provided herein is a method comprising (a) invitro transfecting myeloid progenitor cells with an exogenous genecoding copy of each of the following transcription factors, ERG, HOXA9,and RORA, wherein the transcription factors are expressed in thetransfected cells to produce a resultant population of multilineagehematopoietic progenitor cells (MHPCs) that have myeloid and erythroidpotential; (b) (i) inhibiting a histone methyltransferase in theresultant population of MHPCs to expand lymphoid potential, or (ii) invitro transfecting resultant population of MHPCs with an exogenous genecoding copy of DACH1 and NFIA to expand lymphoid potential, or (iii)both (i) and (ii); and (c) differentiating the resultant population ofMHPCs in the presence of a notch ligand or supportive stroma or both topromote differentiation into the lymphoid lineage.

In another embodiment, this disclosure provides a method of generatingof modified immune cells from a population of myeloid progenitor cellscomprising: (a) in vitro transfecting the myeloid progenitor cells withan exogenous copy of each of the following transcription factors ERG,HOXA9, and RORA, wherein the transfected transcription factors areexpressed in vivo in the cells to produce a population of multilineageprogenitor cells (MHPCs) that having myeloid, and erythroid potential;(b) (i) inhibiting a histone methyltransferase enzyme that targets thehistone protein at H3K9 and/or H3K27 in the resultant population ofMHPCs to expand lymphoid potential, or (ii) in vitro transfectingresultant population of MHPCs with an exogenous gene coding copy ofDACH1 and NFIA to expand lymphoid potential, or (iii) both (i) and (ii);and (c) differentiating the resultant population of MHPCs in thepresence of a notch ligand to promote differentiation into the lymphoidlineage. These immune cells are genetically modified to have exogenouscopies of ERG, HOXA9, and RORA compared to the original myeloidprogenitor cells.

In another embodiment, provided herein is a method comprising (a) invitro contacting or introducing to a population of myeloid progenitorcells a vector or more, the vector(s) collectively carrying an exogenousgene coding copy of each of the following transcription factors, ERG,HOXA9, and RORA, for the in vivo expression of the exogenous copies ofgenes in the contacted cells, wherein the transfected transcriptionfactors are expressed in vivo in the contacted cells to produce apopulation of multilineage hematopoietic progenitor cells (MHPCs) thathaving myeloid and erythroid potential; (b) contacting the MHPCs with aninhibitor of a histone methyltransferase enzyme; and (c) contacting theMHPCs a notch ligand or defined stromal cells or both. In someembodiments, in vitro culturing of the cells occurs between step (a) andstep (b). In some embodiments, the selection of cells occurs betweenstep (a) and step (b). In one embodiment of the method, step (c)consists of activating the Notch signaling pathway in the MHPCs by anymethod known in the art.

In another embodiment, this disclosure provides a method of improving invivo engraftment (also the reconstitution) of hematopoietic stem cellsin a recipient host comprising: (a) in vitro or ex vivo generatingmultilineage hematopoietic progenitor cells (MHPCs) from myeloidprogenitor cells; (b) inhibiting a histone methyltransferase in theresultant population of MHPCs; and (c) transplanting said resultantMHPCs into the recipient host.

In another embodiment, this disclosure provides a modified or engineeredimmune cell produced by a method described herein.

In another embodiment, this disclosure provides a composition comprisingmodified or engineered immune cells produced by a method describedherein.

In another embodiment, this disclosure provides a modified or engineeredimmune cell derived from a population of myeloid progenitor cells,wherein the immune cell comprises an exogenous gene coding copy of eachof the following transcription factors: ERG, HOXA9, and RORA. In oneembodiment, the modified or engineered immune cell further comprises anexogenous gene coding copy of SOX4, or MYB, or both SOX4 and MYB. Inanother embodiment, the modified or engineered immune cell furthercomprises an exogenous gene coding copy of DACH1, or NFIA, or both DACH1and NFIA.

In another embodiment, this disclosure provides a modified or engineeredimmune cell derived from a population of myeloid progenitor cells,wherein the immune cell comprises an exogenous gene coding copy of eachof the following transcription factors: ERG, HOXA9, RORA, DACH1 andNFIA.

In another embodiment, this disclosure provides a modified or engineeredimmune cell derived from a population of myeloid progenitor cells,wherein the immune cell comprises an exogenous gene coding copy of eachof the following transcription factors: ERG, HOXA9, and RORA, and anexogenous gene coding copy of each of the following reprogrammingfactors OCT4, SOX2, KLF4 and optionally c-MYC or NANOG and LIN28, or thefour reprogramming factors: OCT4, SOX2, NANOG and LIN28. In anotherembodiment, the modified cells further comprise an exogenous gene codingcopy of two addition transcription factors, SOX4 and MYB. In anotherembodiment, the modified cells further comprise an exogenous gene codingcopy of two addition transcription factors, DACH1 and NFIA.

In one embodiment, this disclosure provides a composition of modifiedcells derived from a population of myeloid progenitor cells, wherein themodified cell comprises an exogenous copy of each of the followingtranscription factors ERG, HOXA9, and RORA. In another embodiment, themodified cells further comprise an exogenous gene coding copy of twoaddition transcription factors, SOX4 and MYB.

In one embodiment, this disclosure provides a composition of modifiedcells derived from a population of myeloid progenitor cells, wherein themodified cell comprises an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, RORA, SOX4, and MYB.

In one embodiment, this disclosure provides a composition of modifiedcells derived from a population of myeloid progenitor cells, wherein themodified cell comprises an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, RORA, DACH1, NFIA, SOX4, andMYB.

In one embodiment, this disclosure provides a composition of modifiedcells derived from a population of myeloid progenitor cells, wherein themodified cell comprises an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, and RORA, and an exogenousgene coding copy of each of the following reprogramming factors OCT4,SOX2, KLF4 and optionally c-MYC or nanog and LIN28. Alternativecombinations of reprogramming factors include these four factors: OCT4,SOX2, NANOG and LIN28.

In one embodiment, this disclosure provides a composition of modifiedcells derived from a population of myeloid progenitor cells, wherein themodified cell comprises an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, and RORA, SOX4 and MYB, andan exogenous gene coding copy of each of the following reprogrammingfactors OCT4, SOX2, KLF4 and optionally c-MYC or nanog and LIN28.Alternative combinations of reprogramming factors include these fourfactors: OCT4, SOX2, NANOG and LIN28.

In one embodiment of any method, cells or composition described, themyeloid progenitor cells, HPCs, MHPCs, iPSCs, modified or engineeredcell, or modified or engineered immune cell is a mammalian cell. Forexample, the immune cell is a human, rat, mouse, rabbit, or hamstercell.

In one embodiment of any method, cells or composition described, themyeloid progenitor cells, HPCs, MHPCs, iPSCs, modified or engineeredmammalian cell is a primate cell.

In one embodiment of any method, cells or composition described, themyeloid progenitor cells, HPCs, MHPCs, iPSCs, modified or engineeredprimate cell or immune cell is a human cell.

In one embodiment of any method, cells or composition described, theMHPCs are generated by introducing in vitro or ex vivo each of thefollowing transcription factors ERG, HOXA9, and RORA, in the myeloidprogenitor cells, such as the common myeloid progenitor cell (CMP). Forexample, by transfecting with a vector or more, the vector(s)collectively carry an exogenous gene coding copy of each of thefollowing transcription factors, ERG, HOXA9, and RORA, for in vivoexpression of the transcription factors in the transfected cells.

In one embodiment of any method, cells or composition described, theMHPCs are generated by contacting a population of myeloid progenitorcells with a vector or more, wherein the vector(s) collectively carryingan exogenous gene coding copy of each of the following transcriptionfactors, ERG, HOXA9, and RORA, for the in vivo expression of the factorsin the contacted cells, and wherein the transfected transcriptionfactors are expressed in vivo in the contacted cells. For example, afirst vector carrying a nucleic acid sequence of an exogenous genecoding copy of ERG, a second vector carrying a nucleic acid sequence ofan exogenous gene coding copy of HOXA9, and a third vector carrying anucleic acid sequence of an exogenous gene coding copy of RORA.Alternatively, a single vector carrying all the three exogenous genescoding for ERG, HOXA9, and RORA transcription factors.

In one embodiment of any method, cells or composition described, themethod further comprising in vitro transfecting the myeloid progenitorcells with an exogenous gene coding copy of the transcription factors,SOX4, wherein the transfected transcription factor is expressed in vivoin the transfected cells.

In one embodiment of any method, cells or composition described, themethod further comprising in vitro transfecting the myeloid progenitorcells with an exogenous gene coding copy of the transcription factors,MYB, wherein the transfected transcription factor is expressed in vivoin the transfected cells.

In one embodiment of any method, cells or composition described, themyeloid lineage progenitor cells are at least CD45⁺. In one embodimentof any method, cells or composition described, the myeloid lineageprogenitor cells are CD34⁺CD45⁺. In one embodiment of any method, cellsor composition described, the myeloid lineage progenitor cells are atleast CD45⁺ and CD1 lb⁺. In some embodiment, the myeloid lineageprogenitor cells are negative for lymphoid lineage markers such as IL-7R alpha/CD127, CD3, CD4, CD8 and CD19.

In one embodiment of any method, cells or composition described, themyeloid lineage progenitor cells are non-lymphoid lineage committed.

In one embodiment of any method, cells or composition described, theresultant MHPCs are CD34+CD38 negative/low.

In one embodiment of any method, cells or composition described, theresultant MHPCs have myeloid and erythroid but no or very limitedlymphoid potential, less than 5%.

In one embodiment of any method, cells or composition described, themyeloid lineage progenitor cells are progenitor cells are derived fromembryoid bodies obtained from a population of pluripotent stem cells.

In one embodiment of any method, cells or composition described, thepopulation of pluripotent stem cells is iPSCs or embryonic stem cells(ESC).

In one embodiment of any method, cells or composition described, theiPSCs are produced by in vitro or ex vivo introducing exogenous copiesof only three reprogramming factors OCT4, SOX2, and KLF4 into mature orsomatic cells. Alternatively, the iPSC having exogenous copies of thefour reprogramming factors include OCT4, SOX2, NANOG and LIN28.

In one embodiment of any method, cells or composition described, theiPSC having exogenous copies of OCT4, SOX2, and KLF4 is furtherintroduced in vitro or ex vivo with exogenous copies of c-MYC or nanogand LIN28 into the cells.

In one embodiment of any method, cells or composition described, theiPSC are produced by introducing in vitro or ex vivo exogenous copies ofreprogramming factors OCT4, SOX2, and KLF4, and optionally with c-MYC ornanog and LIN28 into mature or somatic cells.

In one embodiment of any method, cells or composition described, theiPSC are produced by in vitro or ex vivo contacting mature cells with avector or more, wherein the vector(s) collectively carry exogenouscopies of reprogramming factors OCT4, SOX2, and KLF4, and optionallywith c-MYC or nanog and LIN28 into mature cells, and wherein thereprogramming factors are expressed in vivo in the contacted mature orsomatic cells.

In one embodiment of any method, cells or composition described, thecells from which iPSC are made can be from any cell type in a donorsubject, any mature or somatic cells. For examples, cells is a bloodsample, or bone marrow sample, B lymphocytes (B-cells), T lymphocytes,(T-cells), fibroblasts, keratinocytes etc.

In one embodiment of any method, cells or composition described, theiPSC are produced by in vitro or ex vivo introducing the disclosedreprogramming factors two or more times into the mature or somaticcells.

In one embodiment of any method, cells or composition described, theiPSC are produced by in vitro or ex vivo contacting mature cells withthe disclosed vector(s) factors two or more times into the mature orsomatic cells.

In one embodiment of any method, cells or composition described, thenotch ligand is Delta-like-1, Delta-like-4, and immobolizedDelta1ext-IgG, consisting of the extracellular domain of humanDelta-like-1 fused to the Fc domain of human IgG1.

In one embodiment of any method, cells or composition described, theDelta-like-1 or Delta-like-4 is supplied with co-culturing the MHPCswith immobolized Delta1ext-IgG, OP9-DL1 cells or OP9-DL4 cells. OP9-DL1cells are a bone-marrow-derived stromal cell line that ectopicallyexpresses the Notch ligand, Delta-like 1 (Dll1).

In one embodiment of any method, cells or composition described, theNotch signaling pathway of the inhibited MHPCs is stimulated in culture.

In one embodiment of any method, cells or composition described, thehistone methyltransferase catalysis the addition of methyl group to thehistone H3 lysine residue 9 (H3K9) and/or histone H3 lysine residue 27(H3K27).

In one embodiment of any method, cells or composition described, thehistone methyltransferase inhibitor inhibits the G9a/GLP heteromericcomplex.

In one embodiment of any method, cells or composition described, thehistone methyltransferase inhibitor inhibits EZH1 (Enhancer Of Zeste 1Polycomb Repressive Complex 2 Subunit).

In one embodiment of any method, cells or composition described, theH3K9 or H3K27 histone methyltransferase is inhibited by a small moleculeor a nucleic acid or a CRISPR-mediated target genetic interference.

In one embodiment of any method, cells or composition described, theH3K27 histone methyltransferase is EZH1.

In one embodiment of any method, cells or composition described, theH3K27 histone methyltransferase is not EZH2.

In one embodiment of any method, cells or composition described, thehistone methyltransferase small molecule inhibitor that is specific toEZH1 and not to EZH2.

In one embodiment of any method, cells or composition described, thehistone methyltransferase small molecule inhibitor include but are notlimited to AMI-1, A-366, BIX-01294, BIX01338, BRD4770, chaetocin,UNCO224, UNC0631, UNC0638, UNC0642, UNC0646, EPZ5676, EPZ005687, GSK343,EPZ-6438, 3-deazaneplanocin A (DZNeP) HCl, UNC1999, MM-102, SGC 0946,Entacapone, EPZ015666, UNC0379, EI1, MI-2 (Menin-MLL Inhibitor), MI-3(Menin-MLL Inhibitor), PFI-2, GSK126, EPZ004777, BRD4770, and EPZ-6438.

In one embodiment of any method, cells or composition described, thehistone methyltransferase nucleic acid inhibitor is a nucleic acidtargeting the expression of the histone methyltransferase.

In one embodiment of any method, cells or composition described, thenucleic acid inhibitor is a RNA interference inhibitor.

In one embodiment of any method, cells or composition described, thenucleic acid is a selected from the group consisting ofCTATCTGGCAGTGCGAGAATG (SEQ. ID. NO: 1), AGACGTGCAAGCAGGTCTTTC (SEQ. ID.NO: 2), TGGATGACTTATGCGTGATTT (SEQ. ID. NO: 3), CAACAGAACTTTATGGTAGAA(SEQ. ID. NO: 4), CCGCCGTGGTTTGTATTCATT (SEQ. ID. NO: 5),GCTTCCTCTTCAACCTCAATA (SEQ. ID. NO: 27), CCGCCGTGGTTTGTATTCATT (SEQ. ID.NO: 28), GCTCTTCTTTGATTACAGGTA (SEQ. ID. NO: 29), andGCTACTCGGAAAGGAAACAAA (SEQ. ID. NO: 30).

In one embodiment of any modified immune cell described, the immune cellfurther comprises an exogenous gene coding copy of SOX4 or MYB or bothSOX4 and MYB.

In one embodiment of any modified immune cell described, the immune cellfurther comprises an exogenous gene coding copy of DACH1 or NFIA or bothDACH1 and NFIA.

In one embodiment of any method, cells or composition described,specific and directed differentiation of the histonemethyltransferase-inhibited MHPCs comprises contacting the cells withcytokines selected from the group consisting of IL-7, IL-2, IL-15, andIL-4.

In one embodiment, provided herein is a method of cellular replacementtherapy, or for the treatment of cancer, autoimmune disorders,hematological diseases, or other genetic diseases and disorders in asubject, comprising (a) providing a somatic cell from a donor subject,(b) generating multilineage hematopoietic progenitor cells from myeloidprogenitor cells derived from the somatic cell as described in any ofthe preceding paragraphs; (c) inhibiting a histone methyltransferase inthe resultant population of multilineage hematopoietic progenitor cellsas described in any of the preceding paragraphs; (d) differentiating theresultant population of multilineage hematopoietic progenitor cells inthe presence of a notch ligand or a stromal cell or both to promotedifferentiation into the lymphoid lineage as described in any of thepreceding paragraphs, and implanting the resultant differentiatedlymphoid cells into a recipient subject.

In one embodiment of the treatment method described above, the hostsubject and the recipient subject are the same individual.

In one embodiment of the treatment method described above, the hostsubject and the recipient subject are not the same individual, but areat least HLA compatible.

Definitions

As used herein, in one embodiment, the term “hematopoietic stem cell” or“HSC” refers to a stem cell that has self-renewal capacity and also giverise to all the blood cell types of the three hematopoietic lineages,erythroid, lymphoid, and myeloid. These cell types include the myeloidlineages (monocytes and macrophages, neutrophils, basophils,eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells),and the lymphoid lineages (T-cells, B-cells, NK-cells). Human HSCs aredetermined as CD34⁺, CD59⁺, CD90/Thy1⁺, CD38^(low/−),c-kit/CD117^(−/low), and Lin⁻. Mouse HSC− are considered CD34^(low/−),SCA-1⁺, CD90/Thy1^(+/low), CD38⁺, c-Kit/CD117⁺, and Lin⁻. Detecting theexpression of these marker panels allows separation of specific cellpopulations via techniques like fluorescence-activated cell sorting(FACS). In one embodiment, the term “hematopoietic stem cell” or “HSC”refers to a stem cell that has self-renewal capacity and that have thefollowing cell surface markers: CD34+, CD59+, Thy1/CD90⁺, CD38^(lo/−),CD133+, c-Kit/CD117^(−/lo), and Lin⁻. In one embodiment, the term“hematopoietic stem cell” or “HSC” refers to a stem cell that is atleast CD34+. In one embodiment, the term “hematopoietic stem cell” or“HSC” refers to a stem cell that has self-renewal capacity and that isat least CD34⁺ and c-kit/CD117^(lo/−). In one embodiment, the term“hematopoietic stem cell” or “HSC” refers to a stem cell that hasself-renewal capacity and that is at least CD38^(low/−),c-kit/CD117^(−/low).

As used herein, the terms “iPS cell”, “iPSC”, and “induced pluripotentstem cell” are used interchangeably and refers to a pluripotent cellartificially derived by the transfection of following reprogrammingfactors OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28, intoa from a differentiated cell, e.g., a somatic cell. Alternativecombinations of reprogramming factors include OCT4, SOX2, NANOG andLIN28.

As used herein, the term “lineage” when used in the context of stem andprogenitor cell differentiation and development refers to the celldifferentiation and development pathway, which the cell can take tobecoming a fully differentiated cell. For example, a HSC has threehematopoietic lineages, erythroid, lymphoid, and myeloid; the HSC hasthe potential, ie., the ability, to differentiate and develop into thoseterminally differentiated cell types known for all these three lineages.When the term “multilineage” used, it means the cell is able to, in thefuture, differentiate and develop into those terminally differentiatedcell types known for more than one lineage. For example, the HSC hasmultilineage potential. When the term “limited lineage” used, it meansthe cell can differentiate and develop into those terminallydifferentiated cell types known for one lineage. For example, a commonmyeloid progenitor cell (CMP) or a megakaryocyte-erythroid progenitor(MEP) has a limited lineage because the cell can only differentiate anddevelop into those terminally differentiated cell types of the myeloidlineage and not that of the lymphoid lineage. Terminally differentiatedcells of the myeloid lineage include erythrocytes, monocytes,macrophages, megakaryocytes, myeloblasts, dendritic cells, andgranulocytes (basophils, neutrophils, eosinophils, and mast cells); andterminally differentiated cells of the lymphoid lineage include Tlymphocytes/T cells, B lymphocytes/B cells, dendritic cells, and naturalkiller cells.

As used herein, the term “a progenitor cell” refers to an immature orundifferentiated cell that has the potential later on to mature(differentiate) into a specific cell type (a fully differentiated orterminally differentiated cell), for example, a blood cell, a skin cell,a bone cell, or hair cells. Progenitor cells have a cellular phenotypethat is more primitive (e.g., is at an earlier step along adevelopmental pathway or progression than is a fully differentiatedcell) relative to a cell, which it can give rise to by differentiation.Often, progenitor cells also have significant or very high proliferativepotential. Progenitor cells can give rise to multiple distinctdifferentiated cell types or to a single differentiated cell type,depending on the developmental pathway and on the environment in whichthe cells develop and differentiate. A progenitor cell also canproliferate to make more progenitor cells that are similarly immature orundifferentiated.

As used herein, the term “multilineage hematopoietic progenitor cells”,“multipotent hematopoietic progenitor cells” and “MHPCs” are usedinterchangeably and refer to hematopoietic cells (cell that form theblood) that have the ability or potential to generate, or differentiateinto, multiple types of hematopoietic lineage cells. In one embodiment,the term includes the “reverse multilineage hematopoietic progenitorcells” “reverse MHPCs” or described herein. Such cells are derived frommyeloid progenitor cells after the in vitro or ex vivo transfection toincorporate several exogenous copies of gene coding nucleic acids of thetranscription factors: ERG, HOXA9, and RORA into the cell. In oneembodiment, the term includes “embryonic body-derived progenitors” and“EB-derived progenitors.”

As used herein, in one embodiment, the term “myeloid progenitor cells”or “myeloid lineage progenitor cells” refer to an immature orundifferentiated cell that is committed to the myeloid lineage and canonly differentiate and develop into those terminally differentiated celltypes of the myeloid lineage. Examples are CMP, MEP, and GMPs of themyeloid lineages. In one embodiment, the term “myeloid progenitor cells”or “myeloid lineage progenitor cells” refer to the CD34+CD45+ cellsderived from embryonic bodies obtained pluripotent stem cells. In oneembodiment, the term “myeloid progenitor cells” or “myeloid lineageprogenitor cells” refer to cells that only differentiate and developinto granulocytes and macrophages.

The term “differentiated cell” is meant any primary cell that is not, inits native form, pluripotent as that term is defined herein. The term a“differentiated cell” also encompasses cells that are partiallydifferentiated, such as multipotent cells (e.g. adult somatic stemcells). In some embodiments, the term “differentiated cell” also refersto a cell of a more specialized cell type derived from a cell of a lessspecialized cell type (e.g., from an undifferentiated cell or areprogrammed cell) where the cell has undergone a cellulardifferentiation process.

In the context of cell ontogeny, the term “differentiate”, or“differentiating” is a relative term meaning a “differentiated cell” isa cell that has progressed further down the developmental pathway thanits precursor cell. Thus in some embodiments, a reprogrammed cell asthis term is defined herein, can differentiate to lineage-restrictedprecursor cells (such as a mesodermal stem cell or a endodermal stemcell), which in turn can differentiate into other types of precursorcells further down the pathway (such as an tissue specific precursor,for example, a cardiomyocyte precursor, or a pancreatic precursoe), andthen to an end-stage differentiated cell, which plays a characteristicrole in a certain tissue type, and may or may not retain the capacity toproliferate further.

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that is able to differentiate into some but not all ofthe cells derived from all three germ layers. Thus, a multipotent cellis a partially differentiated cell. Multipotent cells are well known inthe art, and examples of muiltipotent cells include adult somatic stemcells, such as for example, hematopoietic stem cells and neural stemcells, hair follicle stem cells, liver stem cells etc. Multipotent meansa stem cell may form many types of cells in a given lineage, but notcells of other lineages. For example, a multipotent blood stem cell canform the many different types of blood cells (red, white, platelets,etc. . . . ), but it cannot form neurons; cardiovascular progenitor cell(MICP) differentiation into specific mature cardiac, pacemaker, smoothmuscle, and endothelial cell types; pancreas-derived multipotentprogenitor (PMP) colonies produce cell types of pancreatic lineage(cells that produces insulin, glucagon, amylase or somatostatin) andneural lineage (cells that are morphologically neuron-like,astrocytes-like or oligodendrocyte-like).

The term a “reprogramming gene”, as used herein, refers to a gene whoseexpression, contributes to the reprogramming of a differentiated cell,e.g. a somatic cell to an undifferentiated cell (e.g. a cell of apluripotent state or partially pluripotent state, multipotent state). Areprogramming gene can be, for example, genes encoding mastertranscription factors Sox2, Oct3/4, Klf4, Nanog, Lin-28, c-myc and thelike. The term “reprogramming factor” refers to the protein encoded bythe reprogramming gene.

The term “exogenous” refers to a substance present in a cell other thanits native source. The terms “exogenous” when used herein refers to anucleic acid (e.g. a nucleic acid encoding a reprogramming transcriptionfactor, e.g. Sox2, Oct3/4, Klf4, Nanog, Lin-28, c-myc and the like) or aprotein (e.g., a transcription factor polypeptide) that has beenintroduced by a process involving the hand of man into a biologicalsystem such as a cell or organism in which it is not normally found orin which it is found in lower amounts. A substance (e.g. a nucleic acidencoding a sox2 transcription factor, or a protein, e.g., a SOX2polypeptide) will be considered exogenous if it is introduced into acell or an ancestor of the cell that inherits the substance.

The term “isolated” as used herein signifies that the cells are placedinto conditions other than their natural environment. The term“isolated” does not preclude the later use of these cells thereafter incombinations or mixtures with other cells.

As used herein, the term “expanding” refers to increasing the number oflike cells through cell division (mitosis). The term “proliferating” and“expanding” are used interchangeably.

As used herein, a “cell-surface marker” refers to any molecule that isexpressed on the surface of a cell. Cell-surface expression usuallyrequires that a molecule possesses a transmembrane domain. Somemolecules that are normally not found on the cell-surface can beengineered by recombinant techniques to be expressed on the surface of acell. Many naturally occurring cell-surface markers are termed “CD” or“cluster of differentiation” molecules. Cell-surface markers oftenprovide antigenic determinants to which antibodies can bind to. Acell-surface marker of particular relevance to the methods describedherein is CD34. The useful hematopoietic progenitor cells according tothe present disclosure preferably express DC34 or in other words, theyare CD34 positive.

A cell can be designated “positive” or “negative” for any cell-surfacemarker, and both such designations are useful for the practice of themethods described herein. A cell is considered “positive” for acell-surface marker if it expresses the marker on its cell-surface inamounts sufficient to be detected using methods known to those of skillin the art, such as contacting a cell with an antibody that bindsspecifically to that marker, and subsequently performing flow cytometricanalysis of such a contacted cell to determine whether the antibody isbound the cell. It is to be understood that while a cell may expressmessenger RNA for a cell-surface marker, in order to be consideredpositive for the methods described herein, the cell must express it onits surface. Similarly, a cell is considered “negative” or“negative/low” (abbreviated as “−/lo” or “lo/−”) for a cell-surfacemarker if the cell does not express the marker on its cell surface inamounts sufficient to be detected using methods known to those of skillin the art, such as contacting a cell with an antibody that bindsspecifically to that marker and subsequently performing flow cytometricanalysis of such a contacted cell to determine whether the antibody isbound the cell. In some embodiments, where agents specific forcell-surface lineage markers used, the agents can all comprise the samelabel or tag, such as fluorescent tag, and thus all cells positive forthat label or tag can be excluded or removed, to leave uncontactedhematopoietic stem or progenitor cells for use in the methods describedherein.

As used herein, the term “a histone methyltransferase inhibitor” or“inhibitor” is any molecule that inhibits of expression of a histonemethyltransferase (e.g., G9a, GLP, EZH1), or inhibits the catalyticactivity of the enzyme to methylate lysine resides on the substratehistone protein. For example, a histone methyltransferase inhibitor canbe an siRNA or dsRNA that inhibits of expression of G9a, GLP, or EZH1 inthe inhibited cell, or a gRNA that promotes the degradation of the mRNAof G9a, GLP, or EZH1 in the inhibited cell. For example, a histonemethyltransferase inhibitor is a small molecule that antagonizes theenzyme activity. Examples include but are not limited to small moleculesAMI-1, A-366, BIX-01294, BIX01338, BRD4770, chaetocin, UNCO224, UNC0631,UNC0638, UNC0642, UNC0646, EPZ5676, EPZ005687, GSK343, EPZ-6438,3-deazaneplanocin A (DZNeP) HCl, UNC1999, MM-102, SGC 0946, Entacapone,EPZ015666, UNC0379, EI1, MI-2 (Menin-MLL Inhibitor), MI-3 (Menin-MLLInhibitor), PFI-2, GSK126, EPZ004777, BRD4770, and EPZ-6438 as describedherein.

As used herein, the term “small molecule” refers to a chemical agentincluding, but not limited to, peptides, peptidomimetics, amino acids,amino acid analogs, polynucleotides, polynucleotide analogs, aptamers,nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,including heteroorganic and organometallic compounds) having a molecularweight less than about 10,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 5,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 1,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 500 grams per mole, and salts, esters,and other pharmaceutically acceptable forms of such compounds. In someembodiments, the small molecule is a heterorganic compound or anorganometallic compound.

The term “inhibitory RNA” is meant to include a nucleic acid moleculethat contains a sequence that is complementary to a target nucleic acid(e.g., a target microRNA) that mediates a decrease in the level oractivity of the target nucleic acid. Non-limiting examples of inhibitoryRNAs include interfering RNA, shRNA, siRNA, ribozymes, antagomirs, andantisense oligonucleotides. Methods of making inhibitory RNAs aredescribed herein. Additional methods of making inhibitory RNAs are knownin the art. In one embodiment, the BCL11A microRNA described herein isan inhibitory RNA that causes a decrease in the activity of BCL11A mRNA.

As used herein, “an interfering RNA” refers to any double stranded orsingle stranded RNA sequence, capable—either directly or indirectly(i.e., upon conversion) of inhibiting or down-regulating gene expressionby mediating RNA interference. Interfering RNA includes, but is notlimited to, small interfering RNA (“siRNA”) and small hairpin RNA(“shRNA”). “RNA interference” refers to the selective degradation of asequence-compatible messenger RNA transcript.

As used herein “an shRNA” (small hairpin RNA) refers to an RNA moleculecomprising an antisense region, a loop portion and a sense region,wherein the sense region has complementary nucleotides that base pairwith the antisense region to form a duplex stem. Followingpost-transcriptional processing, the small hairpin RNA is converted intoa small interfering RNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family. As used herein, thephrase “post-transcriptional processing” refers to mRNA processing thatoccurs after transcription and is mediated, for example, by the enzymesDicer and/or Drosha.

A “small interfering RNA” or “siRNA” as used herein refers to any smallRNA molecule capable of inhibiting or down regulating gene expression bymediating RNA interference in a sequence specific manner. The small RNAcan be, for example, about 18 to 21 nucleotides long. Each siRNA duplexis formed by a guide strand and a passenger strand. The endonucleaseArgonaute 2 (Ago 2) catalyzes the unwinding of the siRNA duplex. Onceunwound, the guide strand is incorporated into the RNA InterferenceSpecificity Complex (RISC), while the passenger strand is released. RISCuses the guide strand to find the mRNA that has a complementary sequenceleading to the endonucleolytic cleavage of the target mRNA.

Retroviruses are RNA viruses that utilize reverse transcriptase duringtheir replication cycle. The term “retrovirus” refers to any knownretrovirus (e.g., type c retroviruses, such as Moloney murine sarcomavirus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammarytumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemiavirus (FLV), spumavirus

The retroviral genomic RNA is converted into double-stranded DNA byreverse transcriptase. This double-stranded DNA form of the virus iscapable of being integrated into the chromosome of the infected cell;once integrated, it is referred to as a “provirus.” The provirus servesas a template for RNA polymerase II and directs the expression of RNAmolecules, which encode the structural proteins and enzymes needed toproduce new viral particles.

At each end of the provirus are structures called “long terminalrepeats” or “LTRs.” The term “long terminal repeat (LTR)” refers todomains of base pairs located at the ends of retroviral DNAs which, intheir natural sequence context, are direct repeats and contain U3, R,and U5 regions. LTRs generally provide functions fundamental to theexpression of retroviral genes (e.g., promotion, initiation andpolyadenylation of gene transcripts) and to viral replication. The LTRcontains numerous regulatory signals including transcriptional controlelements, polyadenylation signals and sequences needed for replicationand integration of the viral genome. The viral LTR is divided into threeregions called U3, R and U5. The U3 region contains the enhancer andpromoter elements. The U5 region is the sequence between the primerbinding site and the R region and contains the polyadenylation sequence.The R (repeat) region is flanked by the U3 and U5 regions. The LTRcomposed of U3, R, and U5 regions, appears at both the both the 5′ and3′ ends of the viral genome. In one embodiment of the invention, thepromoter within the LTR, including the 5′ LTR, is replaced with aheterologous promoter. Examples of heterologous promoters that can beused include, for example, a spleen focus-forming virus (SFFV) promoter,a tetracycline-inducible (TET) promoter, a β-globin locus control regionand a β-globin promoter (LCR), and a cytomegalovirus (CMV) promoter.

The term “lentivirus” refers to a group (or genus) of retroviruses thatgive rise to slowly developing disease. Viruses included within thisgroup include HIV (human immunodeficiency virus; including HIV type 1,and HIV type 2), the etiologic agent of the human acquiredimmunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis(visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates. Diseases caused by theseviruses are characterized by a long incubation period and protractedcourse. Usually, the viruses latently infect monocytes and macrophages,from which they spread to other cells. HIV, FIV, and SIV also readilyinfect T lymphocytes, i.e., T-cells.

The term “R region” refers to the region within retroviral LTRsbeginning at the start of the capping group (i.e., the start oftranscription) and ending immediately prior to the start of the poly Atract. The R region is also defined as being flanked by the U3 and U5regions. The R region plays an important role during reversetranscription in permitting the transfer of nascent DNA from one end ofthe genome to the other.

The term “promoter/enhancer” refers to a segment of DNA which containssequences capable of providing both promoter and enhancer functions. Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous,”“exogenous,” or “heterologous.” An “endogenous” enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An“exogenous” or “heterologous” enhancer/promoter is one which is placedin juxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of that gene isdirected by the linked enhancer/promoter.

A “nucleic acid,” as described herein, can be RNA or DNA, and can besingle or double stranded, and can be selected, for example, from agroup including: nucleic acid encoding a protein of interest,oligonucleotides, nucleic acid analogues, for example peptide-nucleicacid (PNA), pseudo-complementary PNA (pc-PNA), and locked nucleic acid(LNA). Such nucleic acid sequences include, for example, but are notlimited to, nucleic acid sequence encoding proteins, for example thatact as transcriptional repressors, antisense molecules, ribozymes, smallinhibitory nucleic acid sequences, for example but are not limited toRNAi, shRNAi, siRNA, microRNAi (miRNA), and antisense oligonucleotides.

As used herein, the term “engraftment” in reference to a recipient hostis when the new blood-forming cells start to grow and which arederivedfrom the implanted cells and make healthy blood stem cells thatshow up in recipient's blood after a minimum period of 10 days afterimplantation. Engraftment can occur as early as 10 days after transplantbut is more common around 14-20 days.

As used herein, the term “reconstitution” with respect to the immunesystem or the blood system in a recipient host refers to the rebuildingthe innate reservoir or working system, or part thereof within the bodyof recipient host to a natural or a functionally state. For example,such as bone marrow after chemotherapy had obliterated the bone marrowstem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F collectively show the in vitro screen for epigeneticmodifiers that restrict definitive lymphoid potential.

FIG. 1A shows the scheme for embryoid body (EB) differentiation of humaniPSC into hematopoietic progenitors. EBs cultured in serum, BMP4 andhematopoietic cytokines were dissociated after 14 days. CD34⁺progenitors were isolated by MACS sorting and transduced with HOXA9,ERG, RORA, SOX4 and MYB in doxycycline (Dox)-inducible lentiviralvectors (5F). 5F cells were then transduced with individual shRNAstargeting each epigenetic modifier, then seeded onto OP9-DL1 stromalco-culture in a 96-well plate to induce T cell differentiation. Dox wasadded to cultures for 20 days to sustain transgene expression and thenremoved thereafter. T cell potential was assessed by flow cytometry onday 35.

FIG. 1B shows the Venn diagram summarizing the candidate hits from twoindependent experiments using two different IPSC lines, CD45-IPS andMSC-IPS. The screen was performed by transduction with 5F followed bysuperinfection of shRNAs, then the transduced cells were co-culturedwith OP-DL1 stroma. The top candidates from the screen are listed. Eachcandidate was scored as a hit if at least 2 of 4 shRNAs producedCD4+CD8+ T cells at higher frequency and higher absolute cell countscompared to control shRNAs targeting luciferase (shLUC).

FIG. 1C shows the relative expansion of 5F+shRNA cells after 14 daysrespecification in +Dox culture.

FIG. 1D shows the prospective analysis of CD4+CD8+ T cell frequenciesfrom 5F+shRNA targeting indicated epigenetic modifier.

FIG. 1E shows the prospective analysis of CD19+ B cell frequencies fromindicated 5F+shRNA cells.

FIG. 1F shows the expansion and differentiation potential of 5F+shEZH1cells after long-term in vitro culture. 5F+shEZH1 cells were maintainedin +Dox cultures for the normal 14 days respecification (˜10²-foldexpansion), plus an additional 6 weeks (˜10⁴-fold expansion) and thenplated into OP9-DL1 stromal coculture. Representative flow cytometricanalyses of T cell potential of 5F+shLUC and 5F+shEZH1 cells afterlong-term culture and differentiation (13 weeks) are shown.

FIGS. 2A-2F collectively show that the repression of EZH1 unlocksmultilymphoid potential with minimal effects on myeloerythroiddifferentiation.

FIG. 2A shows the flow cytometry analysis of CD4+CD8+ T cell developmentof 5F cells with two different shRNAs targeting luciferase (shLUC) orEZH1 (shEZH1). Cells were assessed at 35 days following of co-culturewith OP9-DL1.

FIG. 2B shows that the knockdown of EZH1 robustly promotes B cell(CD19+) potential in iPSC-derived 5F cells as assessed by flowcytometry.

FIG. 2C shows that the myeloid (CD1 lb) cells differentiation are notimpaired in 5F+shEZH1 cells.

FIG. 2D shows that the erythroid (CD71+GLYA+) cells differentiation arenot impaired in 5F+shEZH1 cells.

FIG. 2E shows the quantitation of T cell potential of 5F+shEZH1 cellscompared to 5F+shLUC cells. Graph is shown as mean±SEM of 5 independentreplicates using CD34-iPS, CH45-iPS and MSC-iPS lines. ***p<0.001.

FIG. 2F shows the colony-forming potential of 5F+shLUC or 5F+shEZH1cells plated without Dox. (Top Row) Representative images of CFU-G,CFU-M, CFU-GM, CFU-GEMM and CFU-E colonies on plates without Dox.(Bottom Row, Left) Representiatve images of 5F+shLUC or 5F+shEZH1plates. (Bottom Row, Right) Quantitation of colony-forming potential of5F+shLUC or 5F+shEZH1 cells in two independent experiments (n=2).

FIGS. 3A-3J collectively show that the repression of canonical PRC2subunits does not unlock robust lymphoid potential.

FIG. 3A shows the representative flow cytometry plots of 5F cells witheach indicated PRC2 subunit knocked down using two different shRNAs.

FIG. 3B shows the quantitative PCR of mRNA knockdown efficiency ofindividual shRNAs.

FIG. 3C shows the quantitation of T cell frequencies from 5F plus shRNAtargeting the indicated subunit shown as mean±SEM of two independentexperiments.

FIG. 3D shows the schematic for rescue experiments. 5F cells are GFP+and shRNAs are selectable by puromycin. 5F+shEZH1 cells were transducedwith murine EZH1 ORF (mEzh1) or mEzh1 with the catalytic SET domaindeleted (mEzh1ΔSet), both marked by mCherry fluorescence. GFP+,puro-resistant, mCherry+ cells were sorted and seeded into OP9-DL1stromal co-culture for T cell differentiation.

FIG. 3E shows the representative flow cytometric plots of rescueexperiments detailed in FIG. 3D. All plots are gated on CD45+.

FIG. 3F shows the quantitation of flow cytometric analysis in FIG. 3E,data presented as mean±SEM of two independent experiments.

FIG. 3G shows the dose-dependent decrease in EZH2 and EZH1 enzymaticactivity with increasing concentration of GSK126 as monitored by totalprotein levels of the H3K27me3 in 5F cells. At 3 uM, protein levels oftotal H3K27me3 begins to decrease relative to DMSO control, indicatingeffective dose for EZH2 and EZH1 inhibition.

FIG. 3H shows the flow analysis of T cell potential after treatment ofCD34+d9 hemogenic endothelial (HE) cells without 5F treated with anescalating dose GSK126.

FIG. 3I shows the representative images of colony assays plated with 5Fcells treated with the indicated GSK126 concentration.

FIG. 3J shows the quantitation of colonies in (G) as ±SEM of tworeplicates.

FIGS. 4A-4H collectively show that gene expression and chromatinaccessibility of definitive respecified progenitors.

FIG. 4A shows the 104 genes were significantly upregulated and 49 geneswere significantly downregulated (>2-fold; t-test, p<0.1) upon EZH1knockdown compared to control knockdown in 5F cells.

FIG. 4B shows the GO analysis of the most significantly upregulatedgenes in FIG. 4A.

FIG. 4C shows the GSEA analysis of human HSC and progenitor signaturesin 5F+shEZH1 compared with 5F+shLUC cells. HSC_MLP, MLP and GMPsignatures are significantly enriched (FDR<0.25) in 5F+shEZH1 cells.

FIG. 4D shows the plot of all ATAC-seq peaks in 5F+shEZH1 and 5F+shLUCcells.

FIG. 4E shows the GO analysis of enriched pathways of regions associatedwith upregulated ATAC-seq peaks.

FIG. 4F shows the comparison of genomic regions associated withupregulated ATAC-seq peaks and HSPC, T, B cell GRNs and HSPC signatures.*p<0.05.

FIG. 4G shows the GO analysis of enriched pathways of regions associatedwith downregulated ATAC-seq peaks.

FIG. 4H shows the comparison of genomic regions associated withupregulated ATAC-seq peaks and HSPC, T, B cell GRNs and HSPC signatures.*p<0.05.

FIGS. 5A-5H collectively show that EZH1 directly binds and regulates HSCand lymphoid gene networks.

FIG. 5A shows that the EZH1 or EZH2 tagged with V5 epitope wasoverexpressed in 5F cells and subjected to ChIP-sequencing analysis.ChIP-seq peaks were defined within proximal promoter regions (−1 to +1kb of TSS). EZH1 and EZH2 ChIP-seq peaks were overlapped to identifyunique EZH1-bound promoters.

FIG. 5B is the ChIP-seq density heatmaps for H3K4me3, H3K27me3, EZH1 andEZH2.

FIG. 5C shows the proportion of histone marks associated with EZH1 andEZH2 promoters.

FIG. 5D shows the mRNA expression heatmap of unique EZH1 bound TFs, 152out of 1069 total genes, and their regulated network.

FIG. 5E shows that the significantly upregulated networks of EZH1-boundTFs (FDR<0.25) are enriched in HSPC, B and T cell GRNs.

FIG. 5F shows that EZH1-bound TFs are specifically expressed in HSC, MLPand Pro-B cell populations of the HSPC hierarchy.

FIG. 5G shows that the enrichment of EZH1-bound genes to each populationof HSPC hierarchy (left) and the breakdown of their associated histonemarks (right).

FIG. 5H shows that the EZH1-bound, bivalent genes are highly expressedin B, T, NK, granulocyte and monocyte lineages.

FIGS. 6A-6N collectively show that Ezh1 deficiency increases lymphoidpotential and engraftment of embryonic hematopoietic stem/progenitorcells.

FIG. 6A shows the representative images of E9.5 embryo proper (top) andyolk sac (bottom).

FIG. 6B shows the representative flow plots of T cell analysis from E9.5WT or Ezh1−/− EP and YS. YS and EP were dissociated into single cellsand plated into OP9-DL1 stromal co-culture supplemented with 5 ng/mlIL-7 and 5 ng/mL FLT3. After 12 days of stromal co-culture, cells wereharvested and analyzed for T cell development by the markers CD4 andCD8. All plots are gated on CD45.

FIG. 6C shows the representative flow analysis of TCRγδ and TCRβ from WTor Ezh1−/− EP and YS.

FIG. 6D shows the quantitation of the ratio of CD4+CD8+ T cells or TCRβversus TCRγδ from Ezh1−/− YS compared to WT from three independentexperiments.

FIG. 6E shows the representative images of E10.5 embryos.

FIG. 6F shows the quantitative PCR of each PRC2 subunit in YS and AGMfrom E10.5 WT embryos as mean±SEM of three replicates.

FIG. 6G shows the sublethally-irradiated adult NSG females transplantedintravenously with 3.5 ee of whole E10.5 AGM. Mice were bledretroorbitally every 4 weeks to monitor donor chimerism up to 16 weekspost-transplantation. Each dot represents a single transplant recipient.

FIG. 6H shows the lineage distribution of engrafted mice in FIG. 6G.

FIG. 6I shows the sublethally-irradiated adult NSG females transplantedvia tail vein injections with 5 ee of whole E10.5 YS.

FIG. 6J shows the lineage distribution of engrafted mice in (FIG. 6I).

FIG. 6K shows the whole marrow from primary recipients in FIG. 6Gtransplanted into secondary recipients 24 weeks after primarytransplantation. Two to five primary recipients from each group weresacrificed and 4×10⁶ whole bone marrow cells were transplanted into 1-3secondary recipients.

FIG. 6L shows the lineage distribution of secondary recipients in FIG.6K.

FIG. 6M shows the secondary transplantation of primary recipients inFIG. 6I.

FIG. 6N shows the lineage distribution of secondary recipients in FIG.6M. *p<0.05, ** p<0.01, N.E.=not engrafted.

FIGS. 7A-7B collectively show that the screening for epigeneticmodifiers that can restrict T cell potential.

FIG. 7A shows the of candidate chromatin factors. Four shRNAs targetingeach factor were used in the screen.

FIG. 7B shows the representative flow plots showing T cell potential of5F cells with each top candidate factor knocked down with shRNAs.

FIG. 8 shows the significantly enriched GSEA networks. Statisticallysignificant upregulated or downregulated pathways on day 4, 14 or 28after EZH1 knockdown in 5F cells assessed by RNA sequencing.

FIGS. 9A-9C collectively show the ATAC-sequencing analysis of 5F+shEZH1versus 5F+shLUC cells.

FIG. 9A. GO analysis of enriched pathways of nearest neighbor genesassociated with upregulated ATAC-seq peaks.

FIG. 9B GO analysis of nearest neighbor genes associated withupregulated ATAC peaks.

FIG. 9C. Comparison of upregulated and downregulated ATAC-seq peaks in5F+shEZH1 cells with HSPC, B, T cell GRN and HSPC hierarchy signatures.

FIGS. 10A-11F collectively show the characterization of adultEzh1-deficient mice.

FIG. 10A shows the quantification of LSK SLAM HSCs in adult bone marrow.

FIG. 10B shows the lineage distribution of WT, Ezh1+/− and Ezh1−/− adultmice (8-12 weeks old). n=3 mice per genotype.

FIG. 10C shows the WBC counts in peripheral blood.

FIG. 10D shows the lymphocyte counts in peripheral blood.

FIG. 10E shows the absolute cell numbers in the thymus.

FIG. 10F shows the representative image of two thymuses from WT, Ezh1+/−and Ezh1−/− mice. ****P<0.0001.

FIGS. 11A-11C collectively show the lineage analysis of hematopoieticpopulations in E9.5 YS.

FIG. 11A shows the gating scheme for (embryo proper) EP and (yolk sac)YS.

FIG. 11B shows the representative flow plots of B cells in EP and YSfrom multiple pooled embryos (left) and quantitation from two replicates(right).

FIG. 11C shows the representative flow plots of T cells in EP and YSfrom multiple pooled embryos (left) and quantitation from two replicates(right).

FIGS. 12A-12C collectively show the in vitro B cell differentiationpotential of E9.5 EP and YS.

FIG. 12A shows the representative flow plots of B1 and B2 progenitorfrequencies after 9 days differentiation on OP9-DL stroma.

FIG. 12B shows the representative images of CD19+ B cells (left)isolated from (FIG. 13A) and after 4 days in class switchrecombination-promoting conditions (right).

FIG. 12C shows the flow analysis of class-switch recombinationefficiencies.

FIGS. 13A-13B collectively show that Ezh1-deficient embryonic HSPCscontribute to adult-type lymphopoiesis in vivo.

FIG. 13A shows the representative flow analysis of B1 and B2 progenitorsin the peritoneal cavity of engrafted primary recipients.

FIG. 13B shows the representative flow analysis of TCRβ and TCRγδfrequencies of donor-derived peripheral CD3+ T cells from engraftedprimary recipients.

FIGS. 14A-14D collectively show that the EZH1-regulated networks sharedbetween mouse and human HSPCs.

FIG. 14A shows the 7 significantly upregulated pathways shared betweenall mouse and human Ezh1-deficient HSPCs.

FIG. 14B shows the three significantly downregulated pathways sharedbetween all mouse and human Ezh1-deficient HSPCs.

FIG. 14C shows the number of genes in each GSEA network shared betweenhuman 5F+shEZH1 and mouse HSPCs sorted from indicated tissue/genotype.

FIG. 14D shows the GO analysis of all shared genes in FIG. 5C.

FIG. 15 is a schematic diagram showing an embodiment production ofvarious myeloid, erythroid, and immune cells from pluripotenthematopoietic stem cells.

FIGS. 16A-16F shows that NFIA and DACH1 are for lymphoid developmentfrom hPSCs.

FIG. 16A shows the Venn diagram of 23 candidate transcription factors(TFs) that are specifically expressed in HSCs, downregulated inEB-derived CD34+ progenitors and not induced in ERG, HOXA9,RORA-transduced hematopoietic progenitors.

FIG. 16B shows the new library of 23 TFs and 5F (HOXA9, ERG, RORA, SOX4,MYB) were cloned into doxycycline-inducible lentiviral vectors andtransduced in EB-derived CD34+ hematopoietic progenitors and plated intocolony assays. Colonies were picked and analyzed for TF integration bygenomic PCR using gene-specific primers.

FIG. 16C shows the schematic for assessing lymphoid potential ofEB-CD34+ cells transduced with 28 or 13 TF subset. The 13 TFs (including5F) that were integrated at the highest frequencies were chosen toassess T and B cell potential by stromal co-culture.

FIG. 16D shows the flow cytometic analysis of T cell potential of 28 TFor 13 TF in two different IPS lines (MSC-IPS1, CD34-IPS).

FIG. 16E shows the flow cytometric analysis of B cell potential of 28 TFor 13 TF in two IPS lines. The 13 TFs are sufficient to uncoverT and Bcell potential.

FIG. 16F shows the reductive strategy to determine minimal TFrequirement for multilymphoid potential. Flow cytometric analysis oflymphoid potential of EB-derived CD34+ cells transduced with all 13 TFor with one TF subtracted at a time. In addition to ERG and RORA, NFIAand DACH1 are required for both T and B cell potential.

DETAILED DESCRIPTION

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. It should beunderstood that this disclosures is not limited to the particularmethodology, protocols, and reagents, etc., described herein and as suchcan vary. The terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.

Definitions of common terms in molecular biology can be found in TheMerck Manual of Diagnosis and Therapy, 19th Edition, published by MerckSharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3) or the 2015 digitalonline edition at merckmanuals.com; Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties. Further, unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular.

Unless otherwise stated, the present disclosure was performed usingstandard procedures known to one skilled in the art, for example, inMichael R. Green and Joseph Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,USA (2012); Davis et al., Basic Methods in Molecular Biology, ElsevierScience Publishing, Inc., New York, USA (1986); Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Immunology (CPI) (John E. Coligan, et.al., ed. John Wiley and Sons, Inc.), Current Protocols in Cell Biology(CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.),Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney,Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods(Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barneseditors, Academic Press, 1st edition, 1998), Methods in Molecularbiology, Vol. 180, Transgenesis Techniques by Alan R. Clark editor,second edition, 2002, Humana Press, and Methods in Meolcular Biology,Vo. 203, 2003, Transgenic Mouse, editored by Marten H. Hofker and Janvan Deursen, which are all herein incorporated by reference in theirentireties.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages willmean±1%.

All patents and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

The disclosure described herein, in a preferred embodiment, does notconcern a process for cloning human beings, processes for modifying thegerm line genetic identity of human beings, uses of human embryos forindustrial or commercial purposes or processes for modifying the geneticidentity of animals which are likely to cause them suffering without anysubstantial medical benefit to man or animal, and also animals resultingfrom such processes.

The present disclosure relates to in vitro or ex vivo methods forproducing functional immune cells from progenitor cells that have littleor no lymphoid potential. For example, myeloid progenitor cells have nolymphoid potential and they do not proliferate and differentiate tolymphoid cells such as natural killer lymphocytes, dendritic cells, Tlymphocytes, and B lymphocytes. Myeloid progenitor cells are committedin the myeloid lineage; they undergo further cell division,differentiation and maturation, and produce the following cell types:megakaryocytes, thrombocytes, granulocytes, erythrocytes, mast cells,myeloblast, basophils, neutrophils, eosinophils, monocytes andmacrophages. The functional immune cells derived from non-lymphoidlineage progenitor cells are modified to carry exogenous DNA copies thatencode for certain transcription factors. In one embodiment,patient-specific functional immune cells can be produced according themethods. The cells are functional because they express T- or B-cellspecific markers and also undergone T cell receptor (TCR) generearrangement.

Accordingly, in one embodiment, provided herein is an in vitro or exvivo method comprising (a) generating multilineage hematopoieticprogenitor cells (MHPCs) from myeloid progenitor cells; (b) inhibiting ahistone methyltransferase in the resultant population of MHPCs; and, (c)differentiating the resultant population of MHPCs in the presence of anotch ligand or defined stromal cells or both to promote differentiationinto the lymphoid lineage. In some embodiments, in vitro culturing ofthe cells occurs between the steps. In some embodiments, the cellculturing serves to expand the number of cells of interest at each stepprior to performing the next step of the method. In some embodiments,selection of cells occurs between steps.

In another embodiment, provided herein is a method comprising (a) invitro transfecting myeloid progenitor cells with an exogenous genecoding copy of each of the following transcription factors, ERG, HOXA9,and RORA, wherein the transcription factors are expressed in thetransfected cells to produce a resultant population of MHPCs that haveboth myeloid and erythroid potential; (b) inhibiting a histonemethyltransferase in the resultant population of MHPCs to expandlymphoid potential therein; and (c) differentiating the resultantpopulation of MHPCs in the presence of a notch ligand or supportivestroma or both to promote differentiation into the lymphoid lineage. Insome embodiments, in vitro culturing of the cells occurs between thesteps at each step prior to performing the next step of the method. Insome embodiments, selection of cells occurs between steps. In someembodiments, the culturing serves to expand the number of cells ofinterest.

In another embodiment, this disclosure provides a method of generatingof modified immune cells or modified hematopoietic progenitor cells(HPCs) from a population of myeloid progenitor cells comprising: (a) invitro transfecting the myeloid progenitor cells with an exogenous copyof each of the following transcription factors ERG, HOXA9, and RORA,wherein the transfected transcription factors are expressed in vivo inthe cells to produce a population of MHPCs that having myeloid anderythroid potential; (b) inhibiting a histone methyltransferase thatmethylate histone 3 lysine 9 or lysine 27 residue in the histone (H3K9or H3K27 or both) in the resultant population of MHPCs; and (c)differentiating the resultant population of MHPCs in the presence of anotch ligand to promote differentiation into the lymphoid lineage. Theseimmune cells are genetically modified. In some embodiments, in vitroculturing of the cells occurs between the steps. In some embodiments,selection of cells occurs between steps. In some embodiments, theculturing serves to expand the number of cells of interest at each stepprior to performing the next step of the method.

In another embodiment, provided herein is a method comprising (a)introducing or contacting a population of myeloid progenitor cells witha vector or more, the vector(s) collectively carrying an exogenous genecoding copy of each of the following transcription factors, ERG, HOXA9,and RORA, for in vivo expression in the contacted cells, wherein thetransfected transcription factors are expressed in vivo in the contactedcells to produce a population of MHPCs that having myeloid and erythroidpotential; (b) contacting the MHPCs with an inhibitor of a histonemethyltransferase; and (c) contacting the MHPCs a notch ligand ordefined stromal cells or both. In some embodiments, in vitro culturingof the cells occurs between the steps. In some embodiments, selection ofcells occurs between steps. In some embodiments, the culturing serves toexpand the number of cells of interest at each step prior to performingthe next step of the method.

In another embodiment, this disclosure provides a method of improving invivo engraftment of hematopoietic stem cells (HSCs) or HPCs in arecipient host comprising: (a) generating MHPCs from myeloid progenitorcells; (b) inhibiting a histone methyltransferase in the resultantpopulation of MHPCs; and (c) transplanting said resultant MHPCs into thehost. In some embodiments, in vitro culturing of the cells occursbetween the steps. In some embodiments, selection of cells occursbetween steps. In some embodiments, the culturing serves to expand thenumber of cells of interest at each step prior to performing the nextstep of the method. The myeloid progenitor cells have no or limitedlymphoid potential. In one embodiment, co-culturing the myeloidprogenitor cells in OP9-DL1/4 cells does not produce any CD4⁺/CD8⁺cells.

In another embodiment, this disclosure provides a modified or engineeredimmune cell produced by a method described herein. These immune cellsare genetically modified to have exogenous copies of ERG, HOXA9, andRORA compared to the original myeloid progenitor cells.

In another embodiment, this disclosure provides a composition comprisingengineered immune cells produced by a method described herein. In oneembodiment, the composition further comprises a pharmacologicalacceptable carrier. In one embodiment, the pharmacological acceptablecarrier is not cell culture media.

In one embodiment, this disclosure provides a modified myeloidprogenitor cells having reversed lineage to include increased lymphoidlineage potential.

In one embodiment, this disclosure provides a composition which containthe modified modified myeloid progenitor cells having reversed lineageto include increased lymphoid lineage potential.

In one embodiment, this disclosure provides modified myeloid progenitorcells described herein and compositions thereof for use in themanufacture/production of described modified immune cells.

In one embodiment, this disclosure provides modified myeloid progenitorcells described herein and compositions thereof for use in the cellularreplacement therapy, or for the treatment of cancer, autoimmunedisorders, hematological diseases or other genetic diseases anddisorders.

In one embodiment, this disclosure provides an engineered immune cellderived from a population of myeloid progenitor cells, wherein theimmune cell comprises an exogenous gene coding copy of each of thefollowing transcription factors: ERG, HOXA9, and RORA. In anotherembodiment, the immune cell consists essentially of an exogenous genecoding copy of each of the following transcription factors: ERG, HOXA9,and RORA. In a further embodiment, the immune cell consists of anexogenous gene coding copy of each of the following transcriptionfactors: ERG, HOXA9, and RORA. In one embodiment, the immune cellfurther comprise of an exogenous gene coding copy of followingtranscription factor SOX4 or MYB or both SOX4 and MYB. In oneembodiment, the immune cell further comprise of an exogenous gene codingcopy of following transcription factor DACH1 or NFIA or both DACH1 andNFIA.

In another embodiment, this disclosure provides an engineered immunecell or modified myeloid progenitor cell derived from a population ofmyeloid progenitor cells, wherein the immune cell or modified myeloidprogenitor cell comprises an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, and RORA, and an exogenousgene coding copy of each of the following reprogramming factors OCT4,SOX2, KLF4 and optionally c-MYC or nanog and LIN28. Alternatively, thereprogramming factors are OCT4, SOX2, NANOG and LIN28. In anotherembodiment, the immune cell or modified myeloid progenitor cell consistsessentially of an exogenous gene coding copy of each of the followingtranscription factors: ERG, HOXA9, and RORA, and an exogenous genecoding copy of each of the following reprogramming factors OCT4, SOX2,KLF4 and optionally c-MYC or nanog and LIN28. In a further embodiment,the immune cell or modified myeloid progenitor cell consists of anexogenous gene coding copy of each of the following transcriptionfactors: ERG, HOXA9, and RORA, and an exogenous gene coding copy of eachof the following reprogramming factors OCT4, SOX2, KLF4 and optionallyc-MYC or nanog and LIN28. Alternatively, the reprogramming factorsintroduced into the modified cell are OCT4, SOX2, NANOG and LIN28.

In one embodiment, this disclosure provides a composition of modified orengineered immune cells or modified myeloid progenitor cell derived froma population of myeloid progenitor cells, wherein the modified cellcomprises an exogenous copy of each of the following transcriptionfactors ERG, HOXA9, and RORA. In another embodiment, the modified cellor modified myeloid progenitor cell consists essentially of an exogenousgene coding copy of each of the following transcription factors: ERG,HOXA9, and RORA. In a further embodiment, the modified cell or modifiedmyeloid progenitor cell consists of an exogenous gene coding copy ofeach of the following transcription factors: ERG, HOXA9, and RORA.

In one embodiment, the modified cells described further comprise anexogenous gene coding copy of one or both of two addition transcriptionfactors, SOX4 and MYB. In another embodiment, the modified cells furtherconsists essentially an exogenous gene coding copy of one or both of twoaddition transcription factors, SOX4 and MYB. In a further embodiment,the modified cell consists of an exogenous gene coding copy of twoaddition transcription factors, SOX4 and MYB.

In one embodiment, the modified cells described further comprise anexogenous gene coding copy of one or both of two addition transcriptionfactors, DACH1 and NFIA. In another embodiment, the modified cellsfurther consists essentially an exogenous gene coding copy of twoaddition transcription factors, DACH1 and NFIA. In a further embodiment,the modified cell consists of an exogenous gene coding copy of one orboth of two addition transcription factors, DACH1 and NFIA.

In another embodiment, this disclosure provides a modified or engineeredMHPC produced by a method described herein. In another embodiment, thisdisclosure provides a composition comprising engineered MHPCs producedby a method described herein. The engineered MHPC has exogenous genecoding copy of one or more of the following transcription factors: ERG,HOXA9, RORA, SOX4, MYB, DACH1, NFIA, OCT4, SOX2, KLF4, c-MYC, NANOG andLIN28. Combinations of exogenous transcription or reprogramming factorsin the engineered MHPC include ERG, HOXA9, and RORA; ERG, HOXA9, RORA,SOX4 and MYB; ERG, HOXA9, RORA, DACH1, and NFIA; ERG, HOXA9, RORA, SOX4,MYB, DACH1, and NFIA; ERG, HOXA9, RORA, OCT4, SOX2, KLF4 and optionallyc-MYC or NANOG and LIN28; ERG, HOXA9, RORA, SOX4, MYB, OCT4, SOX2, KLF4and optionally c-MYC or nanog and LIN28; ERG, HOXA9, RORA, SOX4, MYB,DACH1, NFIA, OCT4, SOX2, KLF4 and optionally c-MYC or NANOG and LIN28;ERG, HOXA9, RORA, DACH1, NFIA, OCT4, SOX2, KLF4 and optionally c-MYC ornanog and LIN28; ERG, HOXA9, RORA, DACH1, NFIA, OCT4, SOX2, NANOG andLIN28; ERG, HOXA9, RORA, SOX4, MYB, OCT4, SOX2, NANOG and LIN28; andERG, HOXA9, RORA, DACH1, NFIA, SOX4, MYB, OCT4, SOX2, NANOG and LIN28.In one embodiment, the composition further comprises a pharmacologicalacceptable carrier. In one embodiment, the pharmacological acceptablecarrier is not cell culture media.

In one embodiment, this disclosure provides a composition of modifiedcells derived from a population of myeloid progenitor cells, wherein themodified cell comprises an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, RORA, SOX4 and MYB. Inanother embodiment, the modified cells further consists essentially anexogenous gene coding copy of each of the following transcriptionfactors, ERG, HOXA9, RORA, SOX4 and MYB. In a further embodiment, themodified cell consists of an exogenous gene coding copy of each of thefollowing transcription factors, ERG, HOXA9, RORA, SOX4 and MYB.

In one embodiment, this disclosure provides a composition of modifiedcells derived from a population of myeloid progenitor cells, wherein themodified cell comprises an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, RORA, DACH1 and NFIA. Inanother embodiment, the modified cells further consists essentially anexogenous gene coding copy of each of the following transcriptionfactors, ERG, HOXA9, RORA, DACH1 and NFIA. In a further embodiment, themodified cell consists of an exogenous gene coding copy of each of thefollowing transcription factors, ERG, HOXA9, RORA, DACH1 and NFIA.

In one embodiment, this disclosure provides a composition of modifiedcells derived from a population of myeloid progenitor cells, wherein themodified cell comprises an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, and RORA, and an exogenousgene coding copy of each of the following reprogramming factors OCT4,SOX2, KLF4 and optionally c-MYC or nanog and LIN28. In anotherembodiment, the modified cell consists essentially of an exogenous genecoding copy of each of the following transcription factors: ERG, HOXA9,and RORA, and an exogenous gene coding copy of each of the followingreprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC or nanog andLIN28. In a further embodiment, the modified cell consists of anexogenous gene coding copy of each of the following transcriptionfactors: ERG, HOXA9, and RORA, and an exogenous gene coding copy of eachof the following reprogramming factors OCT4, SOX2, KLF4 and optionallyc-MYC or nanog and LIN28. Alternatively, the combinations of fourreprogramming factors, OCT4, SOX2, NANOG and LIN28, are in the modifiedcell.

In one embodiment, this disclosure provides a composition of modifiedcells derived from a population of myeloid progenitor cells, wherein themodified cell comprises an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, RORA, SOX4 and MYB, and anexogenous gene coding copy of each of the following reprogrammingfactors OCT4, SOX2, KLF4 and optionally c-MYC or nanog and LIN28. Inanother embodiment, the modified cell consists essentially of anexogenous gene coding copy of each of the following transcriptionfactors: ERG, HOXA9, RORA, SOX4 and MYB, and an exogenous gene codingcopy of each of the following reprogramming factors OCT4, SOX2, KLF4 andoptionally c-MYC or nanog and LIN28. In a further embodiment, themodified cell consists of an exogenous gene coding copy of each of thefollowing transcription factors: ERG, HOXA9, RORA, SOX4 and MYB, and anexogenous gene coding copy of each of the following reprogrammingfactors OCT4, SOX2, KLF4 and optionally c-MYC or nanog and LIN28.Alternatively, the combinations of four reprogramming factors, OCT4,SOX2, NANOG and LIN28, are in the modified cell.

In one embodiment, this disclosure provides a composition of modifiedcells derived from a population of myeloid progenitor cells, wherein themodified cell comprises an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, RORA, DACH1 and NFIA, and anexogenous gene coding copy of each of the following reprogrammingfactors OCT4, SOX2, KLF4 and optionally c-MYC or nanog and LIN28. Inanother embodiment, the modified cell consists essentially of anexogenous gene coding copy of each of the following transcriptionfactors: ERG, HOXA9, RORA, DACH1 and NFIA, and an exogenous gene codingcopy of each of the following reprogramming factors OCT4, SOX2, KLF4 andoptionally c-MYC or nanog and LIN28. In a further embodiment, themodified cell consists of an exogenous gene coding copy of each of thefollowing transcription factors: ERG, HOXA9, RORA, DACH1 and NFIA, andan exogenous gene coding copy of each of the following reprogrammingfactors OCT4, SOX2, KLF4 and optionally c-MYC or nanog and LIN28.Alternatively, the combinations of four reprogramming factors, OCT4,SOX2, NANOG and LIN28, are in the modified cell.

In one embodiment, this disclosure provides a composition of modifiedcells derived from a population of myeloid progenitor cells, wherein themodified cell comprises an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, RORA, SOX4, MYB, DACH1 andNFIA, and an exogenous gene coding copy of each of the followingreprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC or nanog andLIN28. In another embodiment, the modified cell consists essentially ofan exogenous gene coding copy of each of the following transcriptionfactors: ERG, HOXA9, RORA, SOX4, MYB, DACH1 and NFIA, and an exogenousgene coding copy of each of the following reprogramming factors OCT4,SOX2, KLF4 and optionally c-MYC or nanog and LIN28. In a furtherembodiment, the modified cell consists of an exogenous gene coding copyof each of the following transcription factors: ERG, HOXA9, RORA, SOX4,MYB, DACH1 and NFIA, and an exogenous gene coding copy of each of thefollowing reprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC ornanog and LIN28. Alternatively, the combinations of four reprogrammingfactors, OCT4, SOX2, NANOG and LIN28, are in the modified cell.

In one embodiment, this disclosure provides a pharmacologicalcomposition comprising modified immune cells described herein and apharmacological acceptable carrier, wherein the modified immune cellcomprises an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, RORA, and optionally each of thefollowing transcription factors SOX4, MYB, DACH1 and NFIA. In oneembodiment, the pharmacological acceptable carrier is not cell culturemedia. In one embodiment, the pharmacological composition is acryopreserved composition comprising at least one cryopreservative agentknown in the art.

Pluripotent stem cells (PSCs) have the potential to give rise to all thesomatic tissues. Directed differentiation of PSCs aims to recapitulateembryonic development to generate patient-matched tissues by specifyingthe three germ layers. A common theme in directed differentiation acrossall germ layers is the propensity of PSCs to give rise to embryonic- andfetal-like cell types, which poses a problem for integration andfunction in an adult recipient. This distinction is particularlystriking in the hematopoietic system, which emerges in temporally andspatially separated waves at during ontogeny (Dzierzak and Speck, 2008).The earliest “primitive” progenitors emerge in the yolk sac at 8.5 dpcand give rise to a limited repertoire of macrophages, megakaryocytes andnucleat-ed erythrocytes (Baron et al 2005, Tavian and Peault 2005,Ferkowicz et al 2005). These early embryonic-like progenitors aregenerally myeloid-based and cannot func-tionally repopulate the bonemarrow of adult recipients. By contrast, “definitive” cells withhematopoietic stem cell (HSC) potential emerge later in arterialendothelium within the aorta-gonad-mesonephros (AGM) and otheranatomical sites (Dzierzak and Speck, 2008). Directed differentiation ofPSCs gives rise to hematopoietic progenitors, which resemble those foundin the yolk sac of the early embryo. These lack functionalreconstitution potential, are biased to myeloid lineages, and expressembryonic globins. Thus, understanding key fate determining mechanismsthat promote development of either primitive or definitive lineages iscritical for specifying HSCs, and other adult-like cell types (e.g., redblood cells) from PSCs.

Activation of Wnt pathway in the early mesoderm, and Notch in hemogenicendothelium, are critical for enhancing definitive potential (Kennedy etal. 2012, Sturgeon et al. 2013, Ditadi et al. 2015). Definitivepotential is marked by B and T lymphopoiesis. While lymphoid activityemerges prior to HSCs (Boiers et al 2013, Yoshimoto et al. 2012,Yoshimoto et al 2011), robust B and T cell potential remains a usefulmarker of definitive fate in vitro. Comprehensive gene expressionprofiling has shed light on the molecular distinctions betweenhematopoietic progenitors throughout ontogeny (Mckinney-Freeman et al.2012, Miranda-Saavedra et al. 2009). Several classes of homeobox (Hox) Aand B cluster genes are expressed in definitive, but not yolk sac cells(Sauvageau et al. 1994, McGrath and Palis 1997). Accordingly,overexpression of HoxB4 is sufficient to generate cells with engraftmentpotential from mouse PSCs. Moreover, HOXA cluster genes enhancehematopoietic commitment from human PSCs (Doulatov et al. 2013,Ramos-Mejia et al. 2015, Dou et al. 2016). Despite these advances,definitive hematopoietic potential of PSCs remains limited.

Epigenetic regulation maintains cell identity during development.Differentiation is marked by progressive silencing of alternativelineage programs by repressive mechanisms, including methylation of DNAand histone residues associated with heterochromatin (Dambacher et al2010). For instance, tri-methylation of histone H3 on lysine 27 (H3K27)by Polycomb repressive complex 2 (PRC2) is required for blooddevelopment (Majewski et al. 2008, Majewski et al. 2010,Mochizuki-Kashio et al. 2011, Hidalgo et al. 2012, Xie et al. 2014,Kinkel et al. 2015, Lee et al. 2015, Ikeda et al. 2016). The inventorstested that primitive and definitive hematopoietic programs areco-excluded by epigenetic mechanisms, similarly to alternative lineagefates. The primitive program that emerges in the yolk sac and duringdirected differentiation of PSCs is cemented by repressive mechanismsthat preclude master transcription factors: SCL, RUNX1, GATA2, HOXA,from activating stem cell and lymphoid genes that characterizedefinitive progenitors. The inventors found that alleviating thisrepression would establish definitive potential from PSCs in vitro andearly embryonic progenitors in vivo. The inventors here report thathaploinsufficient reduction in Polycomb group protein EZH1 enablesmultilymphoid output from PSCs, and emergence of HSCs in sites ofprimitive hematopoiesis in vivo. Thus, EZH1 is a novel regulator ofdefinitive hematopoietic potential in vitro and in vivo.

The object of the present disclosure is to provide a solution to theproblem of a scarcity of HLA-matched HSCs for the in vivo cellularreplacement therapy, treatment of various medical diseases/conditions,and for the in vitro studies of disease modeling, drug screening, andhematological diseases, particularly for HLA-matched HSCs that wouldeventually produce immune cells. Another objective is to enhance theengraftment and reconstitution a transplanted hematopoietic related cellor hematopoietic-derived cells in a subject.

The inventors, by introducing at least three transcription factors, ERG,HOXA9, and RORA, into lineage-restricted myeloid progenitor cells, wereable to reverse the lineage potential of these cells, so that theresultant cells now have the capability to proliferate to produce moreprogeny cells, self-renew to progenitor cells, and also to differentiateinto cell types of more than one lineage. This step provides anothersource of cell type for making lymphoid cells and also erythroid cells.Myeloid progenitor cells are committed to the myeloid lineage forfurther differentiation and maturation, and the myeloid lineage producesthe following cell types: megakaryocytes, thrombocytes, erythrocytes,mast cells, myeloblast, basophils, neutrophils, eosinophils, monocytesand macrophages. The myeloid lineage is different from a lymphoidlineage which produces immune cells such as T and B lymphocytes.

The inventors have shown previously that it is possible to make largebulk amounts of lineage-restricted CD34⁺CD45⁺ myeloid precursor cellsfrom iPSC. (See S. Doulatov, et al. 2013, Cell Stem Cell. 13: 459-470,this reference is incorporated herein in its entirety). Human iPSCs weredifferentiated as embryoid bodies (EBs) in the presence of BMP4 andcytokines, as previously described (Chadwick et al., 2003, Blood,102:906-915). Briefly, iPSC colonies were scraped into non-adherentrotating 10 cm plates. EB media was KO-DMEM+20% FBS (Stem CellTechnologies), 1 mM L-glutamine, 1 mM NEAA, penicillin/streptomycin, 0.1mM β-mercaptoethanol, 200 μg/ml h-transferrin, and 50 μg/ml ascorbicacid. After 24 hrs, media was changed by allowing EBs to settle bygravity, and replaced with EB media supplemented with growth factors: 50ng/ml BMP4 (R&D Systems), 300 ng/ml SCF, 300 ng/ml FLT3, 50 ng/ml G-CSF,20 ng/ml IL-6, 10 ng/ml IL-3 (all Peprotech). Media was changed on day5, and day 10. EBs were dissociated on day 14 by digesting withcollagenase B (Roche) for 2 hrs, followed by treatment with enzyme-freedissociation buffer (Gibco), and filtered through an 80 m filter.Dissociated EBs can be frozen in 10% DMSO, 40% FBS freezing solution.

EBs are three-dimensional aggregates of pluripotent stem cells producedand cultured in vitro in the presence of serum. The EBs then wouldproceed to generate a mixture of primitive and definitive hematopoieticprogenitor cell types. Primitive progenitors equate to those that arisein vivo naturally in the earliest stages of embryonic development,whereas at later stages of maturation the embryonic populations giverise to definitive progenitors cells, which behave similarly to thecells typical of adult hematopoiesis. Lineage-restricted CD34⁺CD45⁺myeloid precursor cells appear at day 10 and are expanded until day 14,and then isolated by cell sorting by CD34⁺CD45⁺ surface markers afterdissociation of the cells aggregated in the EB. These myeloidprogenitors showed robust myeloid colony-forming activity: macrophagecolonies (CFU-M), and granulocyte colony (CFU-G), but produce fewerythroid colonies (CFU-E and BFU-E) or mixed colonies:

granulocyte/erythrocyte/macrophage/megakaryocyte colonies (multilineagemyeloid progenitors: CFU-GEMM), and granulocyte/macrophage colonies(CFU-GM). These lineage-restricted CD34⁺CD45⁺ myeloid precursor cellshad no capacity to proliferate or self-renew in culture in the absenceof serum. These CD34⁺CD45⁺ progenitors in serum free media completelydifferentiate into CD34⁻ cells after 7 days of culture and this isconsistent with loss of clonogenic capacity.

From the bulk produced lineage-restricted CD34⁺CD45⁺ myeloid precursorscells, the inventors showed that it was possible to reverse the myeloidrestricted lineage of these cells and induce cell proliferation andself-renewal capability by expressing three transcription factors, ERG,HOXA9 and RORA, (abbreviated herein as the EAR factors) in these cells.(See S. Doulatov, et al. 2013, Cell Stem Cell. 13: 459-470, thisreference is incorporated herein in its entirety). Briefly, open readingframes encoding the three transcription factors, ERG, HOXA9 and RORAwere cloned into lentiviral vectors using LR Clonase (INVITROGEN™). Twolentiviral vectors were used: pSMAL-GFP (constitutive) and pINDUCER-21(doxycycline-regulated) (Meerbrey et al., 2011, 108:3665-3670, thisreference is incorporated herein in its entirety). Lentiviral particleswere produced by transfecting 293T-17 cells (ATCC) with the3rd-generation packaging plasmids. Virus was harvested 12 and 36 hrsafter transfection and concentrated by ultracentrifugation at 23,000 rpmfor 2 hrs. Constructs were titered by serial dilution on 293T cells.Sorted CD34⁺CD45⁺ progenitors were seeded on fibronectin-coated (10ug/cm²) 96 well plates at a density of 2-5×10⁴ cells per well. Theinfection media was IMDM+20% BIT (StemCell Technologies), 1 mML-glutamine, and 0.1 mM P-mercaptoethanol, with 300 ng/ml SCF, 300 ng/mlFLT3, 50 ng/ml G-CSF, 20 ng/ml IL-6, 10 ng/ml IL-3 (all Peprotech).Lentiviral infections were carried out in a total volume of 150 ul.Following gene transfer, progenitors were cultured in suspension ininfection media supplemented with 50 ng/ml SCF, 50 ng/ml FLT3, 50 ng/mlTPO, 50 ng/ml IL6, and 10 ng/ml IL-3 (all R&D Systems). All experimentswith inducible constructs (including all transplantation experiments),infection media was replaced with StemSpan SFEM (StemCell Technologies).Dox was added at 2 ng/ml (Sigma). Culture media was same as above.Cultures were maintained at a density of <1×10⁶ cells/ml, and media waschanged every 3-4 days. Single lentiviral systems can also be used tointroduce the open reading frames encoding the three transcriptionfactors, ERG, HOXA9 and RORA into the selected myeloid progenitor cells.Single lentiviral expression systems and vector cassettes are known inthe art. For example, as taught in U.S. Pat. No. 8,865,467, the contentsare incorporated herein by reference in its entirety. Alternatively,gene transfer can be performed by episomal vectors. Episomal expressionvector systems are known in the art. For example, as taught in U.S. Pat.Nos. 5,624,820; 5,674,703; 6,339,065; 6,410,314; 6,479,279; 6,797,494;6,808,923; 7,294,505; 7,790,446; 8,703,481; 8,187,836; and 9,068,200,the contents of which are incorporated herein by reference in theirentirety.

The resultant transfected lineage-restricted CD34⁺CD45⁺ myeloidprecursor cells produced a CD34⁺ population of cells after 7 days ofculture in serum-free media and this CD34⁺ population of cells continuedto expand from 7-14 days in culture. The production of a population ofcells in a serum-free media indicates the recovery of the self-renewalcapability of multilineage hematopoietic progenitor cells. Within thisexpanded population of CD34⁺ population of cells are cells that are alsoCD38 negative or low, and are CD90⁺ and CD 49⁺. It is well known thatthe multipotent hematopoietic progenitor cells found in cord blood andhematopoietic stem cells are CD34⁺/CD 38⁻. Therefore, by transfectinglineage-restricted CD34⁺CD45⁺ myeloid precursors cells with EAR, a newpopulation of cells that can proliferate and self-renew in serumfree-media and also exhibit cell surface markers that arecharacteristics of multilineage multipotent hematopoietic progenitorcells instead of the original lineage-restricted CD34⁺CD45⁺ myeloidprecursors cells used for the EAR transfection.

Accordingly, the inventors were able to produce multilineage multipotentCD34⁺/CD 38^(lo/−) hematopoietic progenitor cells fromlineage-restricted CD34⁺CD45⁺ myeloid precursor cells by EARtransfection.

In one embodiment, provided herein are modified myeloid progenitor cellsderived from lineage-restricted CD34⁺CD45⁺ myeloid precursor cells, themodified myeloid progenitor cells have reversed lineage that includeincreased lymphoid lineage potential. In one embodiment, the increasedlymphoid lineage potential is at least 5% compared to prior EARtransfection. In other embodiments, the increased lymphoid lineagepotential is at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or more compared to prior to EAR transfection.

The reversed-lineage hematopoietic progenitor cells have myeloid anderythroid lineage potentials and give rise to myelo-erythroid colonies.Moreover, the inventors found that by expressing two additionaltranscription factors, SOX4 and MYB, in the progenitor cells, the invivo engraftment of the reversed-lineage hematopoietic progenitor cellswas enhanced, and there was an increase in the number of mixedmyelo-erythroid colonies from the reversed-lineage CD34⁺CD45⁺hematopoietic progenitor cells. In one embodiment, the enhanced in vivoengraftment is at least 0.1% compared to in the absence of anyadditional transcription factors selected from the group consisting ofSOX4 and MYB. In other embodiments, the enhanced in vivo engraftment isat least 0.2%, at least 0.5%, at least 1%, at least 2%, at least 3%2 atleast 4%, at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or more compared to in theabsence of any additional transcription factors selected from the groupconsisting of SOX4 and MYB.

In addition, the inventors also found that another two transcriptionfactors, DACH1 and NFIA, enhanced lymphoid potential. In one embodiment,the enhanced in vivo lymphoid potential is at least 0.1% compared to inthe absence of any additional transcription factors selected from thegroup consisting of DACH1 and NFIA. In other embodiments, the enhancedin vivo lymphoid potential is at least 0.2%, at least 0.5%, at least 1%,at least 2%, at least 3%2 at least 4%, at least 5%, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, ormore compared to in the absence of any additional transcription factorsselected from the group consisting of DACH1 and NFIA.

By further inhibiting a histone methyltransferase in these reversedlineage multipotent CD34⁺/CD38^(lo/−) hematopoietic progenitor cells,the inventors were able differentiate these cells into immune cells byco-culturing the reversed lineage multipotent hematopoietic progenitorcells with OP9-DL1/4 cells. The OP9-DL1/4 cells express and secrete theNotch ligand which is a factor known of promoting differentiation ofHSCs to T lymphocytes. The Notch ligand activates the Notch signalingpathway in the histone methyltransferase-inhibited, CD34⁺/CD38^(lo/−)hematopoietic progenitor cells. Normally, in the absence of a histonemethyltransferase inhibitor, the reversed lineage multipotent CD34⁺/CD38^(lo/−) hematopoietic progenitor cells produce about 0-5% colonies orcells with T cell potential when cultured with OP9-DL1/4 cells. Incontrast, with EZH1 knockdown (e.g., by using siRNA or a histonemethyltransferase inhibitor) the frequency of T cell potential increasedto 25-30%, at least a five-fold increase. See FIGS. 2A and 2B. In someembodiments, the term “OP9” cells referenced herein refers to OP9-DL1 orOL9-DL4 cells that secrete Notch ligand that activate the Notchsignaling pathway.

In one embodiment of any method, cells, or composition described herein,the MHPCs exhibit increased frequency of T cell potential compared to inthe absence of a histone methyltransferase inhibitor. In one embodiment,the increased frequency of T cell potential is at least 5% compared toprior to in the absence of a histone methyltransferase inhibitor. Inother embodiments, the increased frequency of T cell potential is atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or more compared to prior to in the absence of a histonemethyltransferase inhibitor.

Moreover, by further incorporating the in vivo expression of two othertranscription factors, SOX4 and MYB, into these cells, engraftment andreconstitution of these cells in vivo is enhanced. The inventors alsofound that by further incorporating the in vivo expression of two othertranscription factors, DACH1 and NFIA, into these cells, the lymphoidpotential of these cells is enhanced. See FIG. 16F.

To understand the gene regulatory networks of definitive lymphoiddevelopment from human pluripotent stem cells (hPSCs), in parallel, theinventors screened additional transcription factors that are known to behighly expressed in hematopoietic stem cells (HSCs). The inventorsselected 23 additional transcription factors (TFs) in addition to HOXA9,ERG, RORA, SOX4 and MYB that were HSC-specific, not induced by HOXA9,ERG, and RORA, and downregulated in CD34⁺ cells differentiated fromembryoid bodies (EBs). The library of 28 transcription factors wereintroduced into CD34⁺ EBs and the EBs were plated used in colony-formingassays. Integration-site analysis by PCR of colonies revealed enrichmentin 13 TFs. To test these libraries prospectively, CD34⁺ EBs weretransduced with the 13 TF subset or 28 TF library and plated the cellsonto stromal co-culture to induce T or B cell differentiation. The 13 TFlibrary was sufficient to induce multilymphoid potential from CD34⁺ EBs.To identify the necessary TFs for lymphoid potential, one TF was removedat a time from the 13 TF cocktail, and T and B cell differentiation wereperformed in the transduced EB. The inventors found that the addition ofNFIA and DACH1, together with HOXA9, ERG, RORA, were required for T andB cell development from hPSCs.

NFI genes function as both positive and regulators of genetranscription. NFIA has been shown to regulate erythrocytic/granulocyticlineage switching (Fazi et al 2005, Starnes et al 2009). DACH1 regulatescell cycle progression of myeloid cells and maintain the colonogenicactivity and block the differentiation of myeloid progenitors (Lee et al2012, Lee et al 2012). However, their roles in hematopoietic stem celland definitive lymphoid development have not been previously explored.Here, the inventors demonstrated that these TFs are part of a regulatorynetwork that is required for lymphoid development from hPSCs.

The advantage of the disclosure protocols is the methods enablesemi-permanent bulk production of desired immune cells or other types ofhematopoietic cells (i.e. cells differentiated from multipotent HSCs)from a variety of types of cell source, from stem cells, hematopoieticprogenitor cells, and mature and differentiated somatic cells, all ofwhich can be readily collected from the patient's body.

The produced engineered immune cells or engineered histonemethyltransferase-inhibited, CD34⁺/CD 38l^(o/−) hematopoietic progenitorcells can be transplanted into a patient for various medical treatmentssuch as immune system reconstruction therapy (e.g., after bone marrowablation) or immunotherapy (e.g., in cancer therapy or autoimmunediseases). One added advantage is that if the donor of the source cellsand recipient of the engineered immune cells are the same person, theproduced engineered immune cells have HLA that are identical to therecipient and this avoids host-graft immune rejection after thetransplantation. For recipient patients that are HLA allogeneic to thedonor person of the source cells, host-graft immune rejection is greatlyreduced.

The produced engineered immune cells or engineered histonemethyltransferase-inhibited, CD34+/CD 38− hematopoietic progenitor cellscan also be cryopreserved till needed in the future.

Currently, bone marrow transplantation is the most established cellularreplacement therapy for a variety of hematological disorders. Thefunctional unit of a bone marrow transplant is the hematopoietic stemcell (HSC), which resides at the apex of a complex cellular hierarchyand replenishes blood development throughout life¹. The scarcity ofHLA-matched HSCs severely limits the ability to carry outtransplantation, disease modeling and drug screening. As such, manystudies have aimed to generate HSCs from alternative sources. Advancesin reprogramming to induced pluripotent stem cells (iPSCs)² has providedaccess to a wide array of patient-specific pluripotent cells, apromising source for disease modeling, drug screens and cellulartherapies. However, the inability to derive engraftable hematopoieticstem and progenitor cells from human pluripotent stem cells (hPSCs) haslimited the characterization of hematological diseases to in vitroassays. Generation of HSCs by directed differentiation has remainedelusive, and there is a need for novel approaches to this problem.

One approach to generate HSCs from hPSCs is to specify HSCs from itsontogenetic precursors. It is now widely accepted that HSCs originatefrom hemogenic endothelium (HE) in the aorta-gonad-mesonephros (AGM)³and arterial endothelium in other anatomical sites. Recent work on thedirected differentiation of HE from hPSCs have provided valuableinsights into some of the signaling pathways that control the emergenceof primitive or definitive populations^(4,5); however, theendothelial-to-hematopoietic transition remains incompletely understoodin human hematopoietic development, making rational interventionchallenging.

An alternative to specifying HSCs from its precursor HE is to start withthe short-lived progenitors and convert them to a stem cell state, astrategy that that is define as “respecification”⁶. Respecificationcombines directed differentiation with transcription-based reprogrammingto re-establish HSC fate. The molecular differences between primaryhuman HSCs and progenitors have been well characterized by geneexpression profiling^(7,8), providing a rational approach to introducestem cell genes back into progenitors. The inventors were able to obtaintransplantable HSC by restoring the HSC transcription factor network inprimitive progenitors derived from hPSCs. The proof-of-principle forthis approach is seminal experiments that demonstrate that HoxB4 canrestore HSC properties in murine primitive progenitors⁹.

For the human system^(10,11), a different set of factors were need torestore hHSC properties in human primitive progenitors due tospecies-specific differences. The inventors tailored transcriptionfactor combinations for hPSCs. The inventors had previously reportedthat five transcription factors: ERG, HOXA9, RORA, SOX4, and MYB(abbreviated as 5F) can convert hPSC-derived myeloid-restrictedprecursors into reversibly immortalized multilineage hematopoieticprogenitors⁶. Doxycycline (Dox)-regulated conditional induction of 5Fexpands and maintains an immature CD34⁺CD38⁻ self-renewing state whileDox withdrawal initiates differentiation. The immature CD34⁺CD38⁻self-renewing state is a hHSC property. These cells are abbreviated asCD34-5F cells. The CD34-5F cells give rise to short-term engraftmentafter transplantation in immunodeficient mice, with erythroidprogenitors undergoing maturation and hemoglobin switching in vivo. Thissystem presents a useful platform for modeling hematological disordersdue to its capacity to generate large numbers of engraftable diseasecells for in vitro and in vivo screens.

Generation of iPSCs by somatic cell reprogramming involves globalepigenetic remodeling, and chromatin-modifying enzymes have beencharacterized as barriers or facilitators of reprogramming^(12,13,14.)Within the hematopoietic system, there are many epigenetic changes thatmediate blood development during ontogeny and differentiation from HSCsto mature progeny. The progression from HSCs to differentiated progenyinvolves coordinated control of gene expression programs leading to theactivation or repression of lineage-specific genes. See FIG. 5 . Theevents that lead to the formation of mature lymphocytes that expressantigen receptors involve regulation of both gene expression and DNArecombination, mainly through the control of chromatin accessibilityl¹⁵.HSC state is controlled by a large number of transcription factors andepigenetic modifiers. The inventors used screening strategies findadditional factors that regulate of the HSC fate. The inventors usedshRNA libraries that repress potential negative regulators of HSC fateto screen for transcription factors and epigenetic modifiers.

Accordingly, in one embodiment of any method, cells, or compositiondescribed herein, the MHPCs are generated by introducing in vitro anexogenous gene coding copy each of the following transcription factors:ERG, HOXA9, and RORA, into the myeloid progenitor cells. In oneembodiment, a vector is used as the transport vehicle to introduce anyof the herein described exogenous gene coding copies into the myeloidprogenitor cells. For example, by transfecting the myeloid progenitorcells with a vector or more, wherein the vector(s) collectively carry anexogenous gene coding copy of each of the following transcriptionfactors, ERG, HOXA9, and RORA, for the in vivo expression of thetranscription factor in the transfected cells. For example, bycontacting the myeloid progenitor cells with a vector or more, whereinthe vector(s) collectively carry an exogenous gene coding copy of eachof the following transcription factors, ERG, HOXA9, and RORA, for the invivo expression of the transcription factor in the contacted cells. Forexample, by contacting the myeloid progenitor cells with a nucleic acidor more, wherein the nucleic acid (s) collectively carry an exogenousgene coding copy of each of the following transcription factors, ERG,HOXA9, and RORA, for the in vivo expression of the transcription factorin the contacted cells. In one embodiment, a single vector is used asthe transport vehicle to introduce the exogeneous gene coding copies ofall three transcription factors, ERG, HOXA9, and RORA into the myeloidprogenitor cells. In one embodiment, one or more episomal vectors areused as the transport vehicle to introduce the exogeneous gene codingcopies of the three transcription factors, ERG, HOXA9, and RORA into themyeloid progenitor cells.

In one embodiment of any method, cells, or composition described herein,the MHPCs are generated by contacting a population of myeloid progenitorcells with a vector or more, wherein the vector(s) collectively carryingan exogenous gene coding copy of each of the following transcriptionfactors, ERG, HOXA9, and RORA, for the in vivo expression of the factorsin the contacted cells, and wherein the transfected transcriptionfactors are expressed in vivo in the contacted cells. The contacting isin vitro or ex vivo. In one embodiment, a single vector is used as thetransport vehicle to introduce the exogeneous gene coding copies of allthree transcription factors, ERG, HOXA9, and RORA into the myeloidprogenitor cells. In one embodiment, one or more episomal vectors areused as the transport vehicle to introduce the exogeneous gene codingcopies of the three transcription factors, ERG, HOXA9, and RORA into themyeloid progenitor cells.

In one embodiment of any method, cells, or composition described herein,the MHPCs are generated by contacting the myeloid progenitor cells witha nucleic acid or more, wherein the nucleic acid (s) collectivelycomprises an exogenous gene coding copy of each of the followingtranscription factors, ERG, HOXA9, and RORA, for the in vivo expressionof the transcription factor in the contacted cells. The contacting is invitro or ex vivo.

In one embodiment of any method, cells, or composition described herein,the contacting of the myeloid progenitor cells with any vector(s),nucleic acid(s) or compositions comprising the vector(s) or nucleicacid(s) described herein occurs in vitro or ex vivo.

In one embodiment of any methods, cells, or composition describedherein, the contacting or introduction is repeated at least once.

In one embodiment of any method, cells, or composition described herein,the method further comprising transfecting the myeloid progenitor cellswith an exogenous gene coding copy of SOX4 or MYB or both SOX4 and MYB,wherein the transfected transcription factor(s) is/are expressed in vivoin the transfected cells. The transfecting is in vitro or ex vivo.

In one embodiment of any method, cells, or composition described herein,the method further comprising transfecting the myeloid progenitor cellswith an exogenous gene coding copy of DACH1 or NFIA or both DACH1 andNFIA, wherein the transfected transcription factor is expressed in vivoin the transfected cells. The transfecting is in vitro or ex vivo.

Transcription Factors

ERG (ETS-related gene) is an oncogene meaning that it encodes a proteinthat typically is mutated in cancer. ERG is a member of the ETS(erythroblast transformation-specific) family of transcription factors.The ERG gene encodes for a protein, also called ERG, that functions as atranscriptional regulator. Genes in the ETS family regulate embryonicdevelopment, cell proliferation, differentiation, angiogenesis,inflammation, and apoptosis. The external idenifications for ERG geneare as follows: HGNC: 3446; Entrez Gene: 2078; Ensembl: ENSG00000157554;OMIM: 165080; UniProtKB: P11308; EMBL: AY204741 mRNA and thecorresponding mRNA translation: AAP41719.1; and GENBANK: AY204742 mRNAand the corresponding mRNA translation: AAP41720.1.

Homeobox protein Hox-A9 is a protein that in humans is encoded by theHOXA9 gene. In vertebrates, the genes encoding the homeobox genes classof transcription factors are found in clusters named A, B, C, and D onfour separate chromosomes. Expression of these proteins is spatially andtemporally regulated during embryonic development. Hox-A9 is part of theA cluster on chromosome 7 and encodes a DNA-binding transcription factorwhich may regulate gene expression, morphogenesis, and differentiation.The external idenifications for HOXA9 gene are as follows: HGNC: 5109;Entrez Gene: 3205; Ensembl: ENSG00000078399; OMIM: 142956; UniProtKB:P31269; EMBL: BT006990 mRNA and the corresponding mRNA translation:AAP35636.1; and GENBANK:AC004080 Genomic DNA.

RAR-related orphan receptor alpha (RORa), also known as NR1F1 (nuclearreceptor subfamily 1, group F, member 1) or RORA is a nuclear receptorthat in humans is encoded by the RORA gene. RORa participates in thetranscriptional regulation of some genes involved in circadian rhythm.This nuclear receptor binds DNA as a monomer to ROR response elements(RORE) containing a single core motif half-site 5′-AGGTCA-3′ preceded bya short A-T-rich sequence. In is a key regulator of embryonicdevelopment, cellular differentiation, immunity, circadian rhythm aswell as lipid, steroid, xenobiotics and glucose metabolism. It isconsidered to have intrinsic transcriptional activity, have some naturalligands like oxysterols that act as agonists (25-hydroxycholesterol) orinverse agonists (7-oxygenated sterols), enhancing or repressing thetranscriptional activity, respectively. It is involved in recruitingdistinct combinations of cofactors to target genes regulatory regions tomodulate their transcriptional expression, depending on the tissue, timeand promoter contexts. It regulates genes involved in photoreceptordevelopment including OPN1SW, OPN1SM and ARR3 and skeletal muscledevelopment with MYOD1. It is required for proper cerebellumdevelopment, regulates SHH gene expression, among others, to inducegranule cells proliferation as well as expression of genes involved incalcium-mediated signal transduction. It regulates the circadianexpression of several clock genes, including CLOCK, ARNTL/BMAL1, NPAS2and CRY1. It competes with NR1D1 for binding to their shared DNAresponse element on some clock genes such as ARNTL/BMAL1, CRY1 and NR1D1itself, resulting in NR1D1-mediated repression or RORA-mediatedactivation of clock genes expression, leading to the circadian patternof clock genes expression. Therefore influences the period length andstability of the clock. It also regulates genes involved in lipidmetabolism such as apolipoproteins APOA1, APOAS, APOC3 and PPARG. Inliver, has specific and redundant functions with RORC as positive ornegative modulator of expression of genes encoding phase I and phase IIproteins involved in the metabolism of lipids, steroids and xenobiotics,such as CYP7B1 and SULT2A1. It induces a rhythmic expression of some ofthese genes. In addition, interplays functionally with NR1H2 and NR1H3for the regulation of genes involved in cholesterol metabolism. It isalso involved in the regulation of hepatic glucose metabolism throughthe modulation of G6PC and PCK1. In adipose tissue, it plays a role asnegative regulator of adipocyte differentiation, probably acting throughdual mechanisms. May suppress CEBPB-dependent adipogenesis throughdirect interaction and PPARG-dependent adipogenesis through competitionfor DNA-binding. Downstream of IL6 and TGFB and synergistically withRORC isoform 2, is implicated in the lineage specification ofuncommitted CD4⁺ T-helper (T(H)) cells into T(H)17 cells, antagonizingthe T(H)1 program. Probably regulates IL17 and IL17F expression on T(H)by binding to the essential enhancer conserved non-coding sequence 2(CNS2) in the IL17-IL17F locus. Involved in hypoxia signaling byinteracting with and activating the transcriptional activity of HIF1A.May inhibit cell growth in response to cellular stress. RORA may exertan anti-inflammatory role by inducing CHUK expression and inhibitingNF-kappa-B signaling. The external idenifications for RORA gene are asfollows: HGNC: 10258; Entrez Gene: 6095; Ensembl: ENSG00000069667; OMIM:600825; UniProtKB: P35398; EMBL: U04899 mRNA and the corresponding mRNA:AAA62660.1; GENBANK: L14611 mRNA and the corresponding mRNA translation:AAA02963.1.

HOX- and ETS-family transcription factors HOXA9 and ERG are inducers ofself-renewal and multilineage potential in hematopoietic progenitorsdifferentiated from hPSCs. RORA is a nuclear receptor that plays a rolein maintaining quiescence of hematopoietic progenitors. The addition ofSOX4 and MYB modulates this network to enable myeloid and erythroidengraftment in vivo.

OCT4, SOX2, KLF4 and c-MYC are the original four transcription factorsidentified to reprogram mouse fibroblasts into iPSCs. These same fourfactors were also sufficient to generate human iPSCs. OCT3/4 and SOX2function as core transcription factors of the pluripotency network byregulating the expression of pluripotency-associated genes. Kruppel-likefactor 4 (KLF4) is a downstream target of LIF-STAT3 signaling in mouseES cells and regulates self-renewal. Human iPSCs can also be generatedusing four alternative factors; OCT4 and SOX2 are required but KLF4 andc-MYC could be replaced with NANOG, a homeobox protein important for themaintenance of pluripotency in both ES cells and early embryos, andLIN28, an RNA binding protein. The combination of OCT4, SOX2, NANOG andLIN28 reprogramming factors have been reported to be also sufficient togenerate human iPSCs.

Transcription factor SOX-4 (SOX4). This intronless gene encodes a memberof the SOX (SRY-related HMG-box) family of transcription factorsinvolved in the regulation of embryonic development and in thedetermination of the cell fate. The encoded protein act as atranscriptional regulator after forming a protein complex with otherproteins, such as syndecan binding protein (syntenin). The protein mayfunction in the apoptosis pathway leading to cell death as well as totumorigenesis and may mediate downstream effects of parathyroid hormone(PTH) and PTH-related protein (PTHrP) in bone development. The externalidenifications for Homo sapiens (Human) SOX4 gene are as follows: HGNC:11200; Entrez Gene: 6659; Ensembl: ENSG00000124766; OMIM: 184430;UniProtKB: Q06945; EMBL: BC072668 mRNA and the corresponding mRNAtranslation: AAH72668.1; GENBANK: X65661 mRNA and the corresponding mRNAtranslation: CAA46612.1.

MYB Proto-Oncogene, Transcription Factor (MYB). This gene encodes aprotein with three HTH DNA-binding domains that functions as atranscription regulator. This protein plays an essential role in theregulation of hematopoiesis. This gene may be aberrently expressed orrearranged or undergo translocation in leukemias and lymphomas, and isconsidered to be an oncogene. The external idenifications for the MYBgene are as follows: HGNC: 7545; Entrez Gene: 4602; Ensembl:ENSG00000118513; OMIM: 189990; UniProtKB: P10242; EMBL: AJ606319 mRNAand the corresponding mRNA translation: CAE55170.1; GENBANK: AJ606320mRNA and the corresponding mRNA translation: CAE55171.1.

NFI genes function as both positive and regulators of genetranscription. Nuclear factor 1 A-type (NFIA) has been shown to regulateerythrocytic/granulocytic lineage switching (Fazi et al 2005, Starnes etal 2009). NFIA has been shown to recognize and bind to the palindromicsequence 5′-TTGGC GCCAA-3′ (SEQ ID NO: 111) present in viral andcellular promoters and in the origin of replication of adenovirus type2. NFIA proteins are individually capable of activating transcriptionand replication. The external idenifications for NFIA gene are asfollows: HGNC: 7784; Entrez Gene: 4774; Ensembl: ENSG00000162599; OMIM:600727; UniProtKB: Q12857; EMBL: AK299579 mRNA and the correspondingmRNA translation: BAG61515.1; GENBANK: AC092784 Genomic DNA.

Dachshund Family Transcription Factor 1 (DACH1). This gene encodes achromatin-associated protein that associates with other DNA-bindingtranscription factors to regulate gene expression and cell fatedetermination during development. The protein contains a Ski domain thatis highly conserved from Drosophila to human. DACH1 regulates cell cycleprogression of myeloid cells and maintain the colonogenic activity andblock the differentiation of myeloid progenitors (Lee et al 2012, Lee etal 2012). The transcription factor is involved in regulation oforganogenesis and may be a regulator of SIX1, SIX6 and probably SIX5.Corepression of precursor cell proliferation in myoblasts by SIX1 isswitched to coactivation through recruitment of EYA3 to the SIX1-DACH1complex. Transcriptional activation seems also to involve theassociation of CREBBP. DACH1 also act as a corepressor of SIX6 inregulating proliferation by directly repressing cyclin-dependent kinaseinhibitors, including the p27Kip1 promoter. Furthermore, DACH1 inhibitsTGF-beta signaling through interaction with SMAD4 and NCOR1, and bindsto chromatin DNA via its DACHbox-N domain. However, their roles inhematopoietic stem cell and definitive lymphoid development have notbeen previously explored. The external idenifications for DACH1 gene asfollows: HGNC: 266; Entrez Gene: 1602; Ensembl: ENSG00000276644; OMIM:603803; UniProtKB: Q9UI36; EMBL: AF356492 mRNA and the correspondingmRNA translation: AAL08487.1.

The cDNA encoding the described and desired transcription factors can becloned by methods known in the art into expression vectors for in vivoexpression in the cells. The expression vectors can be constitutive orinducible vectors. The protein and DNA information for transcriptionfactors can be found in the publically available databases such as theGenBank™ database on the National Institute of Health, the UniProt atthe Protein knowledgebase, and GeneCard database at the WeizmannInstitute for Science. The cDNA clones or plasmids carrying the cDNA canbe purchased at BioCat GmbH, and the lentivirus carrying the cDNAs forexpression can also be purchased at Applied Biological Materials (ABM)Inc.

Accordingly, in one embodiment, provided herein is a population ofmodified myeloid lineage progenitor cells having exogenous gene encodingcopies of the transcription factors ERG, HOXA9, and RORA. In oneembodiment, the modified myeloid lineage progenitor cells furthercomprise an exogenous gene coding copy of SOX4, or MYB, or both SOX4 andMYB. In another embodiment, the modified myeloid lineage progenitorcells further comprise an exogenous gene coding copy of DACH1, or NFIA,or both DACH1 and NFIA. In another embodiment, the modified myeloidlineage progenitor cells further comprise exogenous gene coding copiesof reprogramming factors OCT4, SOX2, and KLF4, and optionally with c-MYCor nanog and LIN28, or the exogenous gene coding copies for fourreprogramming factors consisting of OCT4, SOX2, NANOG, and LIN 28. Inanother embodiment, the modified myeloid lineage progenitor cells can becultured expanded in serum-free media, i.e., the modified myeloidlineage progenitor cells under mitosis and self-renewal in serum-freemedia.

Accordingly, in one embodiment, provided herein is a population ofmodified myeloid lineage progenitor cells having exogenous gene encodingcopies of the transcription factors ERG, HOXA9, and RORA, for use inproducing blood cells, such as immune cells, for medical treatments suchas transplant therapy and cancer immune therapy, or for in vitroresearch purposes described herein. In one embodiment, the modifiedmyeloid lineage progenitor cells further comprise an exogenous genecoding copy of SOX4, or MYB, or both SOX4 and MYB. In anotherembodiment, the modified myeloid lineage progenitor cells furthercomprise an exogenous gene coding copy of DACH1, or NFIA, or both DACH1and NFIA. In another embodiment, the modified myeloid lineage progenitorcells further comprise exogenous gene coding copies of reprogrammingfactors OCT4, SOX2, and KLF4, and optionally with c-MYC or nanog andLIN28, or the exogenous gene coding copies for four reprogrammingfactors consisting of OCT4, SOX2, NANOG, and LIN 28. In anotherembodiment, the modified myeloid lineage progenitor cells can becultured expanded in serum-free media, i.e., the modified myeloidlineage progenitor cells under mitosis and self-renewal in serum-freemedia.

In one embodiment of any method, cells, or composition described herein,the myeloid lineage progenitor cells are progenitor cells derived fromembryoid bodies (EB) obtained from a population of pluripotent stemcells. In one embodiment, the pluripotent stem cells are iPSCs. In oneembodiment, the iPSCs are derived from mature, differentiated, somaticcells.

Accordingly, in one embodiment of any method, cells, or compositiondescribed, the method further comprises providing a population ofpluripotent stem cells (PSCs) for generating the myeloid lineageprogenitor cells. In one embodiment, the PSCs are human cells.

In one embodiment of any method, cells, or composition described, themethod further comprises producing myeloid lineage progenitor cells fromthe population of pluripotent stem cells (PSCs). In one embodiment, thePSCs are human cells.

In one embodiment of any method, cells, or composition described herein,the myeloid lineage progenitor cells are produced by first culturing invitro a population of pluripotent stem cells in bone morphogeneticprotein 4 (BMP4), stem cell factor (SCF), Fms-like tyrosine kinase 3(FLT3/CD135), granulocyte-colony stimulating factor (G-CSF/CSF 3), IL-6,and IL-3 for a period of about 10-21 days to form EB from thepluripotent stem cells, dissociating the EB aggregates of cells intosingle cells, and positively selecting for CD34⁺ and CD45⁺ cells fromthe dissociated cells. The positively selected CD34⁺ and CD45⁺ cells arethe myeloid lineage progenitor cells. In one embodiment, the PSCs arecultured for at least 10 days. In other embodiments, the PSCs arecultured for days, at least 11 days, at least 12 days, at least 13 days,at least 14 days, at least 15 days, at least 16 days, at least 17 days,at least 18 days, at least 19 days, at least 20 days or at least 21days. In one embodiment, the transfected or contacted myeloid progenitorcells are cultured for about 7-21 days. In other embodiment, the PSCsare cultured for about 10-20 days, about 10-18 days, about 10-16 days,about 10-14 days, about 10-12 days, about 11-21 days, 11-20 days, about11-18 days, about 11-16 days, about 11-14 days, about 11-13 days, about11-12 days, about 12-21 days, about 12-20 days, about 12-18 days, about12-16 days, about 12-14 days, about 13-21 days, 13-20 days, about 12-18days, about 13-16 days, about 14-20 days, about 14-18 days, about 14-16days, 10-19 days, about 10-17 days, about 10-15 days, about 10-13 days,about 10-11 days, about 11-19 days, 11-19 days, about 11-17 days, about11-15 days, about 12-19 days, about 12-17 days, about 12-15 days, 12-13days, about 14-21 days, about 13-18 days, about 13-17 days, about 13-15days, about 13-14 days, about 15-21 days, about 15-20 days, about 15-19days, about 15-17 days, about 15-16 days, about 16-21 days, about 16-20days, about 16-19 days, about 16-18 days, about 16-17 days, about 17-21days, about 17-20 days, about 17-19 days, about 17-18 days, about 18-21days, about 18-20 days, about 18-19 days, about 19-21 days, about 19-20days, and about 20-21 days.

In one embodiment of any method, cells, or composition described herein,the myeloid lineage progenitor cells are CD34⁺ and CD45⁺. In otherembodiments of any method, cells, or composition described herein, themyeloid lineage progenitor cells are further CD14 positive, or CD15positive, or CD11b positive or positive for a combination of two orthree of these cell surface CD antigens.

In one embodiment of any method, cells, or composition described herein,the myeloid lineage progenitor cells are non-lymphoid lineage committed.In one embodiment, the myeloid lineage progenitor cells exhibitprimarily (>80% of the CFU of the total CFU is a colony forming assay)the following colony-forming activity in culture: CFU-M and CFU-Gcolonies. In other embodiments, the myeloid lineage progenitor cellsproduce more than 82%, more than 84%, more than 86%, more than 88%, morethan 90%, more than 92%, more than 95%, more than 97%, or more than 99%CFU-M and CFU-G colonies out of the total CFU is a colony forming assay.In one embodiment, the myeloid lineage progenitor cells produce fewCFU-E, BFU-E, CFU-GEMM, and CFU-GM colonies (<20% of the total CFU is acolony forming assay). In one embodiment, the myeloid lineage progenitorcells produce less than 18%, less than 16%, less than 14%, less than12%, less than 10%, less than 8%, less than 6%, less than 4%, or lessthan 2%, CFU-E, BFU-E, CFU-GEMM, and CFU-GM colonies out of the totalCFU is a colony forming assay. In vitro colony forming assay can beperformed by any method known in the art. For example, as taught inTashiro K, et al. 2012, Stem Cell Res. 8:300-311, U.S. Pat. Nos.6,103,522, 6,419,918, 683,854, 7,883,861, 7,989,178 9, 9,273,285. Thesereferences are incorporated herein in their entirety. For example, usinga commercially available kit such as Hematopoietic CFC Assays from CellBiolabs Inc. and The Human Colony Forming Cell (CFC) Assay usingMethylcellulose-based Media from R&D Systems.

In another embodiment of any method, cells, or composition describedherein, the myeloid lineage progenitor cells are harvested fromcollected from peripheral blood, cord blood, chorionic villi, amnioticfluid, placental blood, or bone marrow. Myeloid lineage progenitor cellsthat are CD34⁺ and CD45⁺ cells are positively selected from thesesources.

Peripheral blood progenitor cells (PBPC) have become the preferredsource of hematopoetic progenitor cells for allogeneic and autologoustransplantation because of technical ease of collection and shorter timerequired for engraftment. Traditionally, granulocyte-colony stimulatingfactor (G-CSF) has been used to stimulate more PBPC and release ofhematopoetic progenitor cells from the bone marrow. Although regimensusing G-CSF usually succeed in collecting adequate numbers of PBPC fromhealthy donors, 5%-10% will mobilize stem cells poorly and may requiremultiple large volume apheresis or bone marrow harvesting.

In one embodiment of any method, cells, or composition described herein,the population of pluripotent stem cells is induced pluripotent stemcells (iPSCs) or embryonic stem cells (ESC). IPCS and ESC can beproduced by any method known in the art. Methods of producing iPS cellare known in the art, e.g., U.S. Pat. No. 8,058,065, and U.S. PatentApplication Nos: 20110223669, 20120214243, 20130059386, and 20130183759,all of which are incorporated herein by reference in their entireties.

In one embodiment of any method, cells, or composition described herein,the iPSCs are produced by introducing exogenous copies of only threereprogramming factors OCT4, SOX2, and KLF4 into mature or somatic cells.

In one embodiment of any method, cells, or composition described herein,the iPSCs having exogenous gene coding copies of OCT4, SOX2, and KLF4 isfurther introduced with c-MYC or nanog and LIN28 into mature or somaticcells.

In one embodiment of any method, cells, or composition described herein,the iPSCs are produced by introducing exogenous copies of reprogrammingfactors OCT4, SOX2, and KLF4, and optionally with c-MYC or nanog andLIN28 into mature or somatic cells.

In one embodiment of any method, cells, or composition described herein,the iPSCs are produced by contacting mature cells with a vector or more,wherein the vector(s) collectively carry exogenous gene coding copies ofreprogramming factors OCT4, SOX2, and KLF4, and optionally with c-MYC ornanog and LIN28 into mature or somatic cells, and wherein thereprogramming factors are expressed in vivo in the contacted mature orsomatic cells. The contacting is in vitro or ex vivo.

In one embodiment of any disclosed methods, the iPS cell comprises atleast an exogenous copy of a nucleic acid sequence encoding areprogramming factor selected from the group consisting of genes Oct4(Pou5f1), Sox2, cMyc, Klf4, Nanog, Lin 28 and Glisl. In someembodiments, combinations of reprogramming factors are used. Forexample, a combination of four reprogramming factors consisting of Oct4,Sox2, cMyc, and Klf4, or a combination of four reprogramming factorsconsisting of Oct4, Sox2, Nanog, and Lin 28.

In one embodiment of any method, cells, or composition described herein,the mature cells from which iPS cells are made include any somatic cellssuch as B lymphocytes (B-cells), T lymphocytes, (T-cells), andfibroblasts and keratinocytes.

In one embodiment of any method, cells, or composition described herein,the iPSCs are produced by introducing the disclosed reprogrammingfactors two or more times into the mature or somatic cells.

In one embodiment of any method, cells, or composition described herein,the iPSCs are produced by contacting mature cells with the disclosedvector(s) factors two or more times into the mature/somatic cells.

In some embodiments, in vitro culturing of the cells occur between thestep (a) of generating MHPCs and the step (b) of inhibiting the histonemethyltransferase in the multilineage hematopoietic progenitor cells. Insome embodiments, selection of desired cells occurs between step (a) ofgenerating MHPCs and the step (b) of inhibiting the histonemethyltransferase in the MHPCs.

In one embodiment of any method, cells, or composition described herein,the transfected or contacted myeloid progenitor cells carrying the addedexogenous gene coding copy of the described transcription factors arefurther cultured in vitro for a period of time to expand the number ofcells prior to inhibiting the histone methyltransferase. In oneembodiment of any method, cells, or composition described herein, thetransfected or contacted myeloid progenitor cells are cultured for atleast 7 days. In other embodiments, the transfected or contacted myeloidprogenitor cells are cultured for at least 7 days, at least 8 days, atleast 9 days, at least 10 days, at least 11 days, at least 12 days, atleast 13 days, or at least 14 days. In one embodiment, the transfectedor contacted myeloid progenitor cells are cultured for about 7-21 days.In other embodiment, the transfected or contacted myeloid progenitorcells are cultured for about 7-20 days, about 7-18 days, about 7-16days, about 7-14 days, about 7-12 days, about 7-10 days, 8-20 days,about 8-18 days, about 8-16 days, about 8-14 days, about 8-12 days,about 8-10 days, 9-20 days, about 9-18 days, about 9-16 days, about 9-14days, about 9-12 days, about 9-10 days, 10-20 days, about 10-18 days,about 10-16 days, about 10-14 days, about 10-12 days, 11-20 days, about11-18 days, about 11-16 days, about 11-14 days, about 11-13 days, about11-12 days, about 12-20 days, about 12-18 days, about 12-16 days, about12-14 days, 13-20 days, about 12-18 days, about 13-16 days, about 14-20days, about 14-18 days, and about 14-16 days.

In one embodiment of any method, cells, or composition described herein,the culture expanded myeloid progenitor cells carrying the addedexogenous gene coding copy of the described transcription factors arefurther selected for the presence of cell surface marker CD34 (CD34positive) and for the absence or low expression of cell surface markerCD 38 (CD38 low/negative). In other words, the cells obtained afterculture expansion for a period of time described herein are positivelyselected for CD34 and negatively selected against CD38. The selectedCD34⁺CD38^(lo/−) cells are the reverse lineage MHPCs. Selection can beperformed by any method know, for example, by fluorescence activatedcell sorting (facs) as described in US Patent Publication Nos:20090239235, 20090061513, 20140075593, and U.S. Pat. Nos. 5,985,216,6,455,263, 6,461,813, and 6,897,031. These references are incorporatedherein by reference in their entirety.

In one embodiment of any method, cells, or composition described herein,the MHPCs have myeloid and erythroid potential with low or undetectablelymphoid potential. Lymphoid potential is determined by any method knownin the art, e.g., as taught in the Example Section, or as measuredduring in vitro differentiation protocols or following engraftment inreceptive murine hosts.

In one embodiment of any method, cells, or composition described herein,the MHPCs are CD34⁺CD38 low/negative. In one embodiment of any method,cells, or composition described herein, the MHPCs are CD90 positive orCD49f positive or both. In one embodiment of any method, cells, orcomposition described herein, the MHPCs exhibit increased expression ofthe HSC-specific transcription factors HLF, or NF1B, or HOPX, or HMGA2or RBPMS or combinations thereof compared to prior to the introductionof the EAR into the cell. In one embodiment, the increased expression isat least 5% compared to prior to the introduction of the EAR into thecell. In other embodiments, the increased expression is at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, ormore compared to prior to the introduction of the EAR into the cell.

In some embodiment of any methods, cells, or composition describedherein, the MHPC has at least one of the cell surface markercharacteristic of the human hematopoietic progenitor cells: CD34⁺,CD59⁺, Thy1/CD90⁺, CD38^(lo/−), C-kit/CD117^(lo/−) and Lin⁻. Preferably,the multilineage hematopoietic progenitor cells have several of thesemarkers.

In some embodiment of any methods, cells, or composition describedherein, the MHPCs have the cell surface marker characteristic of theerythroid lineage: CD71 and Ter119.

In some embodiments of any methods, cells, or composition describedherein, the myeloid lineage progenitor cell or the MHPC is selected forthe CD34⁺ surface marker prior to any contacting.

Inhibition of Histone H3 Methylation to Promote and Enhance LymphoidPotential

In the course of these experiments, the inventors discovered thatinhibition of specific histone modifying enzymes targeting H3K9 andH3K27 promotes lymphoid potential of hematopoietic progenitors derivedfrom pluripotent stem cells. The histone modifying enzymes are histonelysine methyltransferases. Post-translational modifications of histoneproteins regulate chromatin compaction, mediate epigenetic regulation oftranscription, and control cellular differentiation in health anddisease. Methylation of histone tails is one of the fundamental eventsof epigenetic signaling. Tri-methylation of lysine 9 of histone H3(H3K9) mediates chromatin recruitment of HP1, heterochromatincondensation and gene silencing. Similarly, methylation of H3K27 andH4K20 are associated with a repressed state of chromatin, whereasexpressed genes are methylated at H3K4, H3K36 and H3K79. Methylation ofH3K9 in humans relies mostly on members of the Suv39 family, namelyEHMT1/GLP, EHMT2/G9a, SUV39H1, SUV39H2, SETDB1 and SETDB2, as well asthen non-Suv39 enzymes PRDM2 and ASH1L (Hong Wu et al., StructuralBiology of Human H3K9 Methyltransferases, 2010, PLoS ONE, 5(2):e8570. Incontrast, the methylation of H3K27 is carry out by the polycombrepressive complex 2 (PRC2).

Di/trimethylation of H3K9 is mainly catalyzed by the conserved SUV39H1/2histone methyltransferases, while the polycomb repressive complex 2(PRC2) ensures di/trimethylation of H3K27 (Rea S, 2000. Nature406:593-599; Margueron R, and Reinberg D. 2011. Nature 469:343-349. PRC2comprises the EZH1/2 catalytic subunit, SUZ12, EED, and RBBP7/4(Margueron R, and Reinberg D, 2011).

While wishing not to be bound by theory, inhibiting the histone lysinemethyltransferases that target H3K9 and H3K27 relieves transcriptionalrepression that results from methylation of histone H3, and therebypromotes gene expression which facilitates cell differentiation.

In one embodiment of any method, cells, or composition described herein,the histone methyltransferase catalyzes the addition of methyl group tothe histone H3 lysine residue 9 (H3K9) and/or histone H3 lysine residue27 (H3K27).

In one embodiment of any method, cells, or composition described, thehistone methyltransferase inhibitor inhibits the G9a/GLP heteromericcomplex.

G9a (EC 2.1.1.43) (UniProtKB: Q96KQ7) is also known as EHMT2,(Euchromatic Histone-Lysine N-Methyltransferase 2), G9A HistoneMethyltransferase and protein G9a.

GLP (EC 2.1.1.43) (UniProtKB: Q9H9B1) is also known as EHMT1(Euchromatic Histone-Lysine N-Methyltransferase 1), G9a-Like Protein 1and GLP1.

In one embodiment of any method, cells, or composition described, thehistone methyltransferase inhibitor inhibits EZH1 (Enhancer Of Zeste 1Polycomb Repressive Complex 2 Subunit).

In one embodiment of any method, cells, or composition described, theH3K27 histone methyltransferase is EZH1 (EC:2.1.1.43) (UniproKBQ92800-1).

In one embodiment of any method, cells, or composition described, theH3K27 histone methyltransferase is not EZH2 (EC:2.1.1.43) (UniproQ15910-1).

In one embodiment of any method, cells, or composition described herein,the inhibitor of histone methyltransferase inhibits the gene expressionor protein catalytic activity of the histone methyltransferase.

In one embodiment of any method, cells, or composition described herein,the histone methyltransferase H3K9 and/or H3K27 is inhibited by a smallmolecule or a nucleic acid or a CRISPR-mediated target geneticinterference.

In one embodiment of any method, cells, or composition described, thehistone methyltransferase small molecule inhibitor is a chemical agentincluding, but not limited to, peptides, peptidomimetics, amino acids,amino acid analogs, polynucleotides, polynucleotide analogs, aptamers,nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,including heteroorganic and organometallic compounds) having a molecularweight less than about 10,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 5,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 1,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 500 grams per mole, and salts, esters,and other pharmaceutically acceptable forms of such compounds. In someembodiments, the small molecule is a heterorganic compound or anorganometallic compound.

In one embodiment of any method, cells, or composition described, thehistone methyltransferase small molecule inhibitor include but are notlimited to AMI-1, A-366, BIX-01294, BIX01338, BRD4770, chaetocin,UNCO224, UNC0631, UNC0638, UNC0642, UNC0646, EPZ5676, EPZ005687, GSK343,EPZ-6438, 3-deazaneplanocin A (DZNeP) HCl, UNC1999, MM-102, SGC 0946,Entacapone, EPZ015666, UNC0379, EI1, MI-2 (Menin-MLL Inhibitor), MI-3(Menin-MLL Inhibitor), PFI-2, GSK126, EPZ004777, BRD4770, and EPZ-6438.

In one embodiment of any method, cells, or composition described, thehistone methyltransferase small molecule inhibitor is selected from thegroup consisting of UNC0631, BRD4770, UNC1999, CPI-360, and BIX 01294.

In one embodiment of any method, cells, or composition described herein,the nucleic acid inhibitor is a nucleic acid targeting the expression ofhistone methyltransferase. For example, targeting the mRNA or primarytranscript of the histone methyltransferase, EZH1, thereby inhibitingprotein expression of the enzyme. Histone-lysine N-methyltransferase akaEnhancer Of Zeste 1 Polycomb Repressive Complex 2 Subunit (EZH1) or EC2.1.1.43, is a component of a noncanonical Polycomb repressive complex-2(PRC2) that mediates methylation of histone H3 (see MIM 602812) lys27(H3K27) and functions in the maintenance of embryonic stem cellpluripotency and plasticity. The external identification for the humanEZH1 gene are as follows: HGNC: 3526; Entrez Gene: 2145; Ensembl:ENSG00000108799; OMIM: 601674; UniProtKB: Q92800; EMBL: AB002386 mRNAand thecorresponding mRNA translation: BAA20842.2; GENBANK: BT009782mRNA and thecorresponding mRNA ranslation: AAP88784.1.

In one embodiment, the nucleic acid inhibitor targets the human EZH1mRNA.

In one embodiment of any method, cells, or composition described herein,the nucleic acid inhibitor is a RNA interference inhibitor orCRISPR-mediated genetic interference inhibitor. The RNA interferenceinhibitor can be designed using the predictor RNAi softwares found atthe Whitehead Institute, MIT, sirna website, BLOCK-iT™ RNAi Designer atInvitrogen/ThermoFisher, and other online siRNA design tools at The RNAiWeb using the mRNA of EZH1 as the target.

Similarly, Crisper guide RNA can be designed using the Broad Institute(MIT) crispr software (see MIT website), dna20, Clontech, AddGene,e-crisp, and innovativegenomic using the mRNA or genomic gene of EZH1 asthe target.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)Cas9-mediated gene disruption has been widely used in generatingloss-of-function mutations in diverse organisms including mammals (Conget al., 2013, Science, 339(6121):819-23; reviewed in Hsu et al., 2014,Cell, 157(6):1262-78)). Cas9-based knockout screens have been applied inidentifying essential genes and genes involved in drug resistance invarious cell lines. With respect to general information on CRISPR-CasSystems, components thereof, and delivery of such components, includingmethods, materials, delivery vehicles, vectors, particles, AAV, andmaking and using thereof, including as to amounts and formulations, alluseful in the practice of the instant invention, reference is made to:U.S. Pat. Nos. 8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616,8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965,8,771,945 and 8,697,359; US Patent Publications US 2014-0310830, US2014-0287938, US 2014-0273234, U52014-0273232, US 2014-0273231, US2014-0256046, US 2014-0248702, US 2014-0242700, US 2014-0242699, US2014-0242664, US 2014-0234972, US 2014-0227787, US 2014-0189896, US2014-0186958, US 2014-0186919, US 2014-0186843, US 2014-0179770 and US2014-0179006, US 2014-0170753; European Patents EP 2 784 162 B1 and EP 2771 468 B1; European Patent Applications EP 2 771 468 (EP13818570.7), EP2 764 103 (EP13824232.6), and EP 2 784 162 (EP14170383.5); and PCTPatent Publications WO 2014/093661, all of which are incorporated hereinby reference in their entirety.

The CRISPR/Cas system envisaged for use in the context of the inventioncan make use of any suitable CRISPR enzyme. In some embodiments, theCRISPR enzyme is a type II CRISPR system enzyme. In some embodiments,the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzymeis S. pneumoniae, S. pyogenes, or S. thermophilus Cas9, and may includemutated Cas9 derived from these organisms. The enzyme may be a Cas9homolog or ortholog. In some embodiments, the CRISPR enzyme iscodon-optimized for expression in a eukaryotic cell.

As described herein, the CRISPR/Cas system is used to specificallytarget a multitude of sequences within the continuous genomic region ofinterest. The targeting typically comprises introducing into each cellof a population of cells a vector system of one or more vectorscomprising an engineered, non-naturally occurring CRISPR-Cas systemcomprising: at least one Cas protein, and one or more guide RNAs of theguide RNA library described herein.

In these methods, the Cas protein and the one or more guide RNAs may beon the same or on different vectors of the system and are integratedinto each cell, whereby each guide sequence targets a sequence withinthe continuous genomic region in each cell in the population of cells.The Cas protein is operably linked to a regulatory element to ensureexpression in said cell, more particularly a promoter suitable forexpression in the cell of the cell population. In particularembodiments, the promoter is an inducible promoter, such as adoxycycline inducible promoter. When transcribed within the cells of thecell population, the guide RNA comprising the guide sequence directssequence-specific binding of a CRISPR-Cas system to a target sequence inthe continuous genomic region. Typically binding of the CRISPR-Cassystem induces cleavage of the continuous genomic region by the Casprotein.

RNA interference (RNAi) mediated by short interfering RNAs (siRNA) ormicroRNAs (miRNA) is a powerful method for post-transcriptionalregulation of gene expression. RNAi has been extensively used for thestudy of biological processes in mammalian cells and could constitute atherapeutic approach to human diseases in which selective modulation ofgene expression would be desirable. Depending on the degree ofcomplementarity between miRNA and target mRNA sequences, loss of geneexpression occurs by inducing degradation of the cognate mRNA or bytranslational attenuation. Endogenous miRNAs are transcribed as primarytranscripts and subsequently processed by the RNAse III enzyme Drosha,(1) to create a stem loop structure. Nuclear export and cleavage byDicer generates a mature short double stranded molecule (siRNA) that isseparated into guide and passenger strands. The guide strand is loadedinto the RNA induced silencing complex (RISC), the effector complexmediating cleavage of target mRNAs with the functional guide strandbinding to RISC proteins while the passenger strand is degraded. Theloading of guide versus passenger strands into RISC largely depends onthe 5′ end stability of the siRNA, with the less stable strandpreferentially incorporated into RISC, although the exact regulation inmammalian cells is incompletely understood. The 5′ end of the guidestrand contains the “seed region,” which is critical for targetidentification. Precise cleavage by Drosha and Dicer is critical for thegeneration of guide RNAs with defined seed regions that mediateefficient binding to the appropriate target mRNAs. Inaccurate processingresults in binding to off-target molecules but a shift in cleavage sitesalso alters the nucleotide composition of duplex ends, which may have aprofound effect on strand loading into RISC.

The inhibiting the expression of selected target polypeptides is throughthe use of RNA interference agents. RNA interference (RNAi) uses smallinterfering RNA (siRNA) duplexes that target the messenger RNA encodingthe target polypeptide for selective degradation. siRNA-dependentpost-transcriptional silencing of gene expression involves cleaving thetarget messenger RNA molecule at a site guided by the siRNA. RNAi is anevolutionally conserved process whereby the expression or introductionof RNA of a sequence that is identical or highly similar to a targetgene results in the sequence specific degradation or specificpost-transcriptional gene silencing (PTGS) of messenger RNA (mRNA)transcribed from that targeted gene (see Coburn, G. and Cullen, B.(2002) J. Virology 76(18):9225), thereby inhibiting expression of thetarget gene. In one embodiment, the RNA is double stranded RNA (dsRNA).This process has been described in plants, invertebrates, and mammaliancells. In nature, RNAi is initiated by the dsRNA-specific endonucleaseDicer, which promotes processive cleavage of long dsRNA intodouble-stranded fragments termed siRNAs. siRNAs are incorporated into aprotein complex (termed “RNA induced silencing complex,” or “RISC”) thatrecognizes and cleaves target mRNAs. RNAi can also be initiated byintroducing nucleic acid molecules, e.g., synthetic siRNAs or RNAinterfering agents, to inhibit or silence the expression of targetgenes. As used herein, “inhibition of target gene expression” includesany decrease in expression or protein activity or level of the targetgene or protein encoded by the target gene as compared to a situationwherein no RNA interference has been induced. The decrease will be of atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or more as compared to the expression of a target gene or theactivity or level of the protein encoded by a target gene which has notbeen targeted by an RNA interfering agent.

The terms “RNA interference agent” and “RNA interference” as they areused herein are intended to encompass those forms of gene silencingmediated by double-stranded RNA, regardless of whether the RNAinterfering agent comprises an siRNA, miRNA, shRNA or otherdouble-stranded RNA molecule. siRNA is defined as an RNA agent whichfunctions to inhibit expression of a target gene, e.g., by RNAi. AnsiRNA may be chemically synthesized, may be produced by in vitrotranscription, or may be produced within a host cell. In one embodiment,siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40nucleotides in length, preferably about 15 to about 28 nucleotides, morepreferably about 19 to about 25 nucleotides in length, and morepreferably about 19, 20, 21, 22, or 23 nucleotides in length, and maycontain a 3′ and/or 5′ overhang on each strand having a length of about0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang isindependent between the two strands, i.e., the length of the overhang onone strand is not dependent on the length of the overhang on the secondstrand. Preferably the siRNA is capable of promoting RNA interferencethrough degradation or specific post-transcriptional gene silencing(PTGS) of the target messenger RNA (mRNA).

siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs).In one embodiment, these shRNAs are composed of a short (e.g., about 19to about 25 nucleotide) antisense strand, followed by a nucleotide loopof about 5 to about 9 nucleotides, and the analogous sense strand.Alternatively, the sense strand may precede the nucleotide loopstructure and the antisense strand may follow. These shRNAs may becontained in plasmids, retroviruses, and lentiviruses and expressedfrom, for example, the pol III U6 promoter, or another promoter (see,e.g., Stewart, et al. (2003) RNA April; 9(4):493-501, incorporated byreference herein in its entirety). The target gene or sequence of theRNA interfering agent may be a cellular gene or genomic sequence, e.g.,the BCL11A sequence. An siRNA may be substantially homologous to thetarget gene or genomic sequence, or a fragment thereof. As used in thiscontext, the term “homologous” is defined as being substantiallyidentical, sufficiently complementary, or similar to the target mRNA, ora fragment thereof, to effect RNA interference of the target. Inaddition to native RNA molecules, RNA suitable for inhibiting orinterfering with the expression of a target sequence include RNAderivatives and analogs. Preferably, the siRNA is identical to itstarget. The siRNA preferably targets only one sequence. Each of the RNAinterfering agents, such as siRNAs, can be screened for potentialoff-target effects by, for example, expression profiling. Such methodsare known to one skilled in the art and are described, for example, inJackson et al. Nature Biotechnology 6:635-637, 2003. In addition toexpression profiling, one may also screen the potential target sequencesfor similar sequences in the sequence databases to identify potentialsequences which may have off-target effects. For example, 15, or perhapsas few as 11 contiguous nucleotides, of sequence identity are sufficientto direct silencing of non-targeted transcripts. Therefore, one mayinitially screen the proposed siRNAs to avoid potential off-targetsilencing using the sequence identity analysis by any known sequencecomparison methods, such as BLAST. siRNA sequences are chosen tomaximize the uptake of the antisense (guide) strand of the siRNA intoRISC and thereby maximize the ability of RISC to target G9a/GLP or EZH1mRNA for degradation. This can be accomplished by scanning for sequencesthat have the lowest free energy of binding at the 5′-terminus of theantisense strand. The lower free energy leads to an enhancement of theunwinding of the 5′-end of the antisense strand of the siRNA duplex,thereby ensuring that the antisense strand will be taken up by RISC anddirect the sequence-specific cleavage of the human G9a/GLP or EZH1 mRNA.siRNA molecules need not be limited to those molecules containing onlyRNA, but, for example, further encompasses chemically modifiednucleotides and non-nucleotides, and also include molecules wherein aribose sugar molecule is substituted for another sugar molecule or amolecule which performs a similar function. Moreover, a non-naturallinkage between nucleotide residues can be used, such as aphosphorothioate linkage. The RNA strand can be derivatized with areactive functional group of a reporter group, such as a fluorophore.Particularly useful derivatives are modified at a terminus or termini ofan RNA strand, typically the 3′ terminus of the sense strand. Forexample, the 2′-hydroxyl at the 3′ terminus can be readily andselectively derivatizes with a variety of groups. Other useful RNAderivatives incorporate nucleotides having modified carbohydratemoieties, such as 2′O-alkylated residues or 2′-O-methyl ribosylderivatives and 2′-O-fluoro ribosyl derivatives. The RNA bases may alsobe modified. Any modified base useful for inhibiting or interfering withthe expression of a target sequence may be used. For example,halogenated bases, such as 5-bromouracil and 5-iodouracil can beincorporated. The bases may also be alkylated, for example,7-methylguanosine can be incorporated in place of a guanosine residue.Non-natural bases that yield successful inhibition can also beincorporated. The most preferred siRNA modifications include2′-deoxy-2′-fluorouridine or locked nucleic acid (LAN) nucleotides andRNA duplexes containing either phosphodiester or varying numbers ofphosphorothioate linkages. Such modifications are known to one skilledin the art and are described, for example, in Braasch et al.,Biochemistry, 42: 7967-7975, 2003. Most of the useful modifications tothe siRNA molecules can be introduced using chemistries established forantisense oligonucleotide technology. Preferably, the modificationsinvolve minimal 2′-O-methyl modification, preferably excluding suchmodification. Modifications also preferably exclude modifications of thefree 5′-hydroxyl groups of the siRNA. The Examples herein providespecific examples of RNA interfering agents, such as shRNA moleculesthat effectively target mRNA.

In one embodiment, the nucleic acid is a G9a/GLP or EZH1 specific RNAinterference agent or a vector encoding the RNA interference agent. Inone embodiment, the RNA interference agent comprises one or more of thenucleotide sequences selected from the group consisting ofCTATCTGGCAGTGCGAGAATG (SEQ. ID. NO: 1), AGACGTGCAAGCAGGTCTTTC (SEQ. ID.NO: 2), TGGATGACTTATGCGTGATTT (SEQ. ID. NO: 3), CAACAGAACTTTATGGTAGAA(SEQ. ID. NO: 4), CCGCCGTGGTTTGTATTCATT (SEQ. ID. NO: 5),GCTTCCTCTTCAACCTCAATA (SEQ. ID. NO: 27), CCGCCGTGGTTTGTATTCATT (SEQ. ID.NO: 28), GCTCTTCTTTGATTACAGGTA (SEQ. ID. NO: 29), andGCTACTCGGAAAGGAAACAAA (SEQ. ID. NO: 30).

In one embodiment of any method, cells, or composition described herein,the nucleic acid is selected from the group consisting ofCTATCTGGCAGTGCGAGAATG (SEQ. ID. NO: 1), AGACGTGCAAGCAGGTCTTTC (SEQ. ID.NO: 2), TGGATGACTTATGCGTGATTT (SEQ. ID. NO: 3), CAACAGAACTTTATGGTAGAA(SEQ. ID. NO: 4), CCGCCGTGGTTTGTATTCATT (SEQ. ID. NO: 5),GCTTCCTCTTCAACCTCAATA (SEQ. ID. NO: 27), CCGCCGTGGTTTGTATTCATT (SEQ. ID.NO: 28), GCTCTTCTTTGATTACAGGTA (SEQ. ID. NO: 29), andGCTACTCGGAAAGGAAACAAA (SEQ. ID. NO: 30).

In one embodiment of any method, cells, or composition described herein,the multilineage hematopodetic progenitor cells are contacted with theviral vector or vector carrying a nucleic acid molecule comprising anucleic acid sequence selected from a group consisting of SEQ IDNOS:1-5, 27-30.

In one embodiment of any method, cells, or composition described herein,the contacting with the histone methyltransferase inhibitor occurs morethan once. For example, after the initial first contacting of themultilineage hematopodetic progenitor cell with the virus or vectorcarrying a nucleic acid molecule comprising a nucleic acid sequenceselected from a group consisting of SEQ ID NOS:1-5, 27-30, or contactingwith a small molecule inhibitor described herein, the contacted cell iswashed, and the washed cell is then contacted for a second time with thesame histone methyltransferase inhibitor used in the first contact.

It is contemplated herein that the Cas9/CRISPR system of genome editingbe employed with the methods, cells and compositions described herein.Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated (Cas) systems is useful for RNA-programmablegenome editing (see e.g., Jinek, M. et al. Science (2012)337(6096):816-821).

Trans-activating crRNA (tracrRNA) is a small trans-encoded RNA. It wasfirst discovered in the human pathogen Streptococcus pyogenes. (SeeDeltcheva E, et al. (2011). Nature 471 (7340): 602-7). In bacteria andarchaea, CRISPR/Cas (clustered, regularly interspaced short palindromicrepeats/CRISPR-associated proteins) constitute an RNA-mediated defensesystem which protects against viruses and plasmids. This defensivepathway has three steps. First a copy of the invading nucleic acid isintegrated into the CRISPR locus. Next, CRISPR RNAs (crRNAs) aretranscribed from this CRISPR locus. The crRNAs are then incorporatedinto effector complexes, where the crRNA guides the complex to theinvading nucleic acid and the Cas proteins degrade this nucleic acid.(See Terns M P and Terns R M (2011). Curr Opin Microbiol 14 (3): 321-7).There are several pathways of CRISPR activation, one of which requires atracrRNA which plays a role in the maturation of crRNA. TracrRNA iscomplementary to and base pairs with a pre-crRNA forming an RNA duplex.This is cleaved by RNase III, an RNA-specific ribonuclease, to form acrRNA/tracrRNA hybrid. This hybrid acts as a guide for the endonucleaseCas9, which cleaves the invading nucleic acid. (see Deltcheva E, et al.supra; Jinek M, et al. (2012), Science 337 (6096): 816-21; and Brouns SJ (2012), Science 337 (6096): 808-9).

In some embodiments, Cas9/CRISPR system guide RNAs are designed totarget the exon 3 of EZH1 gene, which is present in all transcripts ofEZH1 known. Exon 3 sequence isATTACAGCAAGATGGAAATACCAAATCCCCCTACCTCCAAATGTATCACTTACTGGAAAAGAAAAGTGAAATCTGAATACATGCGACTTCGACAACTTAAACGGCTTCAGGCAAATATGGGT GCAAAG(SEQ ID NO:6).

Non-limiting exemplary gRNAs that target exon 3 are TCGACAACTTAAACGGCTTC(SEQ ID NO:7), TGCGACTTCGACAACTTAAA (SEQ ID NO:8), CCTCCAAATGTATCACTTAC(SEQ ID NO:9), TAAACGGCTTCAGGCAAATA (SEQ ID NO:10) AAACGGCTTCAGGCAAATAT(SEQ ID NO:11), CATTTGGAGGTAGGGGGATT (SEQ ID NO:12),CCAGTAAGTGATACATTTGG (SEQ ID NO:13), GTGATACATTTGGAGGTAGG (SEQ IDNO:14), AAGTGATACATTTGGAGGTA (SEQ ID NO:15), AGTGATACATTTGGAGGTAG (SEQID NO:16), TTTCCAGTAAGTGATACATT (SEQ ID NO:17), and TAAGTGATACATTTGGAGGT(SEQ ID NO:18)

In other embodiments, Cas9/CRISPR system guide RNAs are designed totarget the exon 4 of EZH1 gene, which is also present in all transcriptsof EZH1 known. Exon 4 sequence isGCTTTGTATGTGGCAAATTTTGCAAAGGTTCAAGAAAAAACCCAGATCCTCAATGAAGAATGGAAGAAGCTTCGTGTCCAACCTGTTCAGTCAATGAAGCCTGTGAGTGGACACCCTTTTCTC AAAAAG(SEQ ID NO:19).

Non-limiting exemplary gRNAs that target exon 4 are GCTTCATTGACTGAACAGGT(SEQ ID NO:20), ACAGGCTTCATTGACTGAAC (SEQ ID NO:21),AGAAAAGGGTGTCCACTCAC (SEQ ID NO:22), TCCATTCTTCATTGAGGATC (SEQ IDNO:23), CCATTCTTCATTGAGGATCT (SEQ ID NO:24), CCCAGATCCTCAATGAAGAA (SEQID NO:110), GTATGTGGCAAATTTTGCAA (SEQ ID NO:25), andCAGTCAATGAAGCCTGTGAG (SEQ ID NO:26).

In one embodiment of any method, cells, or composition described herein,a vector is used as a transport vehicle to introduce any of the hereindescribed exogenous gene coding copies of transcription factors orreprogramming factors or nucleic acid inhibitor into the target cellsselected from the disclosed myeloid progenitor cells or the disclosedreverse lineage multipotent hematopoietic progenitor cell.

In one embodiment of any method, cells, or composition described herein,a vector is used as a transport vehicle to introduce any of the hereindescribed nucleic acid comprising the described exogenous gene codingcopies of transcription factors or reprogramming factors or nucleic acidinhibitor into the target cells selected from the disclosed myeloidprogenitor cells or the disclosed reverse lineage multipotenthematopoietic progenitor cell.

In one aspect, the present specification provides a vector or more,wherein the vector(s) collectively comprises an exogenous gene codingcopies of each of the transcription factors or reprogramming factors ornucleic acid inhibitor described. The exogenous gene coding copy is forthe expression of the transcription factors or reprogramming factorsinside the cells. The in vivo expression of the nucleic acid inhibitoris for degrading the mRNA of the targeted histone methyltransferase suchas G9a/GLP or EZH1 so as to reduce and inhibit the expression of therespective histone methyltransferase, with the goal being to reducemethylation of the histone H3 in the transfected cells and reliefrepression of gene expression therein. In one embodiment, each vectorconsists essentially of a transcription factors or reprogramming factordescribed herein. In one embodiment, each vector consists essentially oftwo or more of the described transcription factors or reprogrammingfactors.

In one aspect, the present specification provides a vector or more,wherein the vector(s) collectively comprises nucleic acids comprisingthe described exogenous gene coding copies of transcription factors orreprogramming factors or nucleic acid inhibitor. The nucleic acid is forthe expression of the transcription factors or reprogramming factorsinside the cells.

In one aspect, the present specification provides a vector or more,wherein the vector(s) collectively comprises an exogenous gene codingcopy of each of the following transcription factors, ERG, HOXA9, andRORA described herein. For example, a single vector carrying the codingcopies for all three transcription factors, ERG, HOXA9, and RORA. Inanother aspect, the vector(s) collectively further comprise an exogenousgene coding copy of SOX4 and MYB. For example, a single vector carryingthe coding copies for both SOX4 and MYB. In another aspect, thevector(s) collectively further comprise an exogenous gene coding copy ofDACH1 and NFIA. For example, a single vector carrying the coding copiesfor both DACH1 and NFIA.

In another aspect, the present disclosure also provides a host cellcomprising a vector or more described herein or nucleic acid(s) of thetranscription factors or reprogramming factors or both described herein.

In another aspect, the disclosure herein also provides a host cellcomprising a vector or more described herein or nucleic acid(s) of thetranscription factors, ERG, HOXA9, and RORA described herein.

In another aspect, the host cell further comprises a vector or moredescribed herein or nucleic acid(s) of the transcription factors SOX4and MYB.

In another aspect, the host cell further comprises a vector or moredescribed herein or nucleic acid(s) of reprogramming factors or bothdescribed herein, OCT4, SOX2, and KLF4, and optionally with c-MYC orNANOG and LIN28, or the four reprogramming factors OCT4, SOX2, NANOG andLIN28

In one embodiment of any host cell described herein, the host cell is anembryonic stem cell, a somatic stem cell, a progenitor cell, a bonemarrow cell, a hematopoietic stem cell, a hematopoietic progenitor cell,an immune cell such as a T cell or B cell, an erythrocyte, a fibroblast,a keratinocyte, or a myeloid progenitor cell. In one embodiment, thehost cell is isolated from a subject. In one embodiment, the host cellis isolated from a subject who has been diagnosed with a hematologicaldisease.

In one embodiment of any method, cells, or composition described herein,the vector further comprises a spleen focus-forming virus promoter, atetracycline-inducible promoter, a Doxycycline (Dox)-inducible, or aβ-globin locus control region and a β-globin promoter. In oneembodiment, the promoter provide for targeted expression of the nucleicacid molecule therein. Other examples of promoters include but are notlimited to the CMV promoter and EF1α promoters for the varioustransgenes, and U6 promoter for shRNAs targeting EZH1.

In one embodiment of any method, cells, or composition described herein,the vector is a virus or a non-viral vector. Non-limiting examples ofviral vectors for gene delivery and expressions in cells are retrovirus,adenovirus (types 2 and 5), adeno-associated virus (AAV),Helper-dependent adenoviral vector (HdAd), hybrid adenoviral vectors,herpes virus, pox virus, human foamy virus (HFV), and lentivirus.

In one embodiment of any method, cells, or composition described herein,the vector is an episomal vector.

In one embodiment of any method, cells, or composition described herein,the vector is an integrating vector.

In one embodiment of any method, cells, or composition described herein,the vector is a non-integrating vector.

In one embodiment of any method, cells, or composition described herein,the vector is an excisable vector.

In one embodiment of any method, cells, or composition described herein,the in vivo expression of the described transcription factors areregulatable. That is, the promoters used in the vectors for geneexpression are inducible.

In one aspect of any method, cells, or composition described herein, thelentivirus is selected from the group consisting of: humanimmunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type2 (HIV-2), caprine arthritis-encephalitis virus (CAEV), equineinfectious anemia virus (EIAV), feline immunodeficiency virus (FIV),bovine immune deficiency virus (BIV), and simian immunodeficiency virus(SIV).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional nucleic acid segments canbe ligated. Another type of vector is a viral vector, wherein additionalnucleic acid segments can be ligated into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”,or more simply “expression vectors.” In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the methods and compositions described herein can include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, lentiviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions.

Within an expression vector, “operably linked” is intended to mean thatthe nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in atarget cell when the vector is introduced into the target cell). Theterm “regulatory sequence” is intended to include promoters, enhancersand other expression control elements (e.g., polyadenylation signals).Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences).Furthermore, the DNA-targeting endonuclease can be delivered by way of avector comprising a regulatory sequence to direct synthesis of theDNA-targeting endonuclease at specific intervals, or over a specifictime period. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the target cell, the level of expression desired, and the like.

Suitable viral vectors include, but are not limited to, vectors based onRNA viruses, such as retrovirus-derived vectors (for example, Moloneymurine leukemia virus (MLV)-derived vectors), and more complexretrovirus-derived vectors (such as Lentivirus-derived vectors); andvectors based on DNA viruses, such as adenovirus-based vectors andadeno-associated virus (AAV)-based vectors. In some embodiments, thepolynucleotide delivery system comprises a retroviral vector, morepreferably a lentiviral vector. Non-limiting examples of viral vectorinclude lentivirus vectors derived from human immunodeficiency virus 1(HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectiousanemia virus, simian immunodeficiency virus (SIV) and maedi/visna virus.

Induction of Lymphoid Potential and Lymphocyte Differentiation

In another embodiment, this disclosure provides an immune cell producedby a method described herein. These immune cells are geneticallymodified to have exogenous copies of ERG, HOXA9, and RORA compared tothe original myeloid progenitor cells. These immune cells can alsofurther have exogenous copies of SOX4, and MYB compared to the originalmyeloid progenitor cells. These immune cells can also further haveexogenous copies of DACH1 and NFIA compared to the original myeloidprogenitor cells.

In another embodiment, this disclosure provides an immune cell derivedfrom a population of myeloid progenitor cells, wherein the immune cellcomprises an exogenous copy of each of the following transcriptionfactors ERG, HOXA9, and RORA.

In another embodiment, this disclosure provides an immune cell derivedfrom a population of myeloid progenitor cells, wherein the immune cellcomprises an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, and RORA, and an exogenous gene codingcopy of each of the following reprogramming factors OCT4, SOX2, KLF4 andoptionally c-MYC or NANOG and LIN28, or the four reprogramming factorsOCT4, SOX2, NANOG and LIN28.

In one embodiment of any immune cell described, the immune cell furthercomprises an exogenous gene coding copy of SOX4 or MYB or both SOX4 andMYB.

In one embodiment of any immune cell described, the immune cell furthercomprises an exogenous gene coding copy of DACH1 or NFIA or both DACH1and NFIA. DACH1 and NFIA enhance lymphoid potential in the reverselineage MHPCs described herein.

In one embodiment of any immune cell described, the immune cell is aB-cell or a T-cell. In one embodiment of any immune cell described, theT-cell is a T regulatory (T_(Reg)) cell. In one embodiment of any immunecell described, the T-cell is a natural killer cell.

The reverse lineage multipotent hematopoietic progenitor cells areimmortalized and they represent a useful platform amenable to furthergenetic modification such as removal of the native T cell receptor locusto enhance targeted specificity, deletion of class I and class II majorhistocompatibility complexes, and expression of non-canonical HLA-G andHLA-E to prevent NK cell-mediated lysis (Riolobos L et al. 2013), whichcan provide a source of universal T cells for immunotherapy, e.g.,cancer immune therapy. In one embodiment of any immune cell described,the immune cell can undergo further genetic modification to editendogenous HLA (please see Riolobos L et al. 2013), or the removalendogenous TCR for targeted specificity, chimeric antigen receptor (CAR)knock-in.

The reverse lineage MHPCs also retained their lymphoid potential afterlong term in vitro culture, producing ˜10⁸ T cells from an average of˜10⁴ EB cells after 13 weeks of expansion and differentiation. See FIG.1F.

In nature, the haematopoietic stem cells (HSCs) in the bone marrow giverise to multipotent progenitors (MPPs) before differentiating intocommon myeloid progenitors (CMPs) and common lymphoid progenitors(CLPs). CLPs migrate from the bone marrow to the thymus, where thymicepithelial cells that express Delta-like ligand 4 (DLL4) triggercanonical Notch 1 signalling in early thymic progenitors (ETPs). ThisNotch 1 signal is essential for T cell lineage commitment and is furtherrequired during early phases of thymocyte differentiation up to thedouble-negative 3 (DN3) stage. Active Notch signaling during these earlystages of T cell development inhibits other lineage potentials, such asB cell and myeloid cell (including dendritic cell (DC)) potential.During β-selection, Notch signaling is turned off as a consequence ofpre-T cell receptor signaling. Thus subsequent stages of T celldevelopment exhibit very low levels of Notch signaling. Notch was alsosuggested to influence the development of regulatory T (T_(Reg)) cells(specifically, thymic T_(Reg) cells). Notch signaling is mediated by theNotch 2 receptor. Notch signaling pathway is highly conserved in bothvertebrate and invertebrate species and it regulates many different cellfate decisions. It is important for pattern formation during developmentsuch as neurogenesis, angiogenesis or myogenesis and regulates T celldevelopment and stem cell maintenance. Notch signaling is also involvedin cellular processes throughout adulthood. Signaling via Notch occursbetween neighbouring cells and both the receptor and its ligands aretransmembrane proteins. Schmitt T. M., Zúñiga-Pflücker J. C. (2002)Induction of T cell development from hematopoietic progenitor cells bydelta-like-1 in vitro. Immunity 17:749-756; Mohtashami M. (2010) DirectComparison of D111- and D114-Mediated Notch Activation Levels ShowsDifferential Lymphomyeloid Lineage Commitment Outcomes. J Immunol.185(2):867-76; Ohishi K et al. Delta-1 enhances marrow and thymusrepopulating ability of human CD34(⁺)CD380 cord blood cells. J ClinInvest. 2002 October; 110(8):1165-74; and Dallas M H et al. Density ofthe Notch ligand Delta1 determines generation of B and T cell precursorsfrom hematopoietic stem cells J Exp Med. 2005 May 2; 201(9): 1361-1366.

Accordingly, to initiate differentiation in the lymphoid lineage and Tcell lineage commitment in the histone methyltransferase inhibitedmultipotent hematopoietic progenitor cells, these cells are exposed to aNotch ligand to activate the Notch signaling pathway therein.

Notch ligands are single-pass transmembrane proteins with a DSL (Delta,Serrate, LAG-2)-domain and varying numbers of EGF-like repeats. Thereare two classes of canonical Notch ligands, the Delta/Delta-like and theSerrate/Jagged class. The later has an additional domain of cysteinerich repeats close to the transmembrane domain. There are 5 canonicalNotch ligands in mammals: Jagged-1, Jagged-2, DLL1, DLL3 and DLL4. Thesecan bind to the four Notch receptors Notch 1-4. DLL1, also known asNotch Delta ligand, Delta-like 1, is a protein which interacts with aNOTCH2 receptor. Shimizu K, et al., 2001, J. Biol. Chem. 276 (28):25753-8; Blaumueller C M, et al., 1997, Cell 90 (2): 281-91; Shimizu K,et al., 2000, Mol. Cell. Biol. 20 (18): 6913-22. DLL1 is a protein thatin humans is encoded by the DLL1 gene. DLL1 is a human homolog of theNotch Delta ligand.

There are several ways to provide a Notch ligand. These include but arenot limited to co-culturing with stroma cells such as OP-9-DL1 orsimilar cells that express and display extracellular or secretes such aNotch ligand, and by providing a purified recombinant form of a Notchligand or a Notch receptor-binding fragment, the receptor-bindingfragment being sufficient to elicit cell signaling events in vivo uponcontact and binding with the extracellular Notch receptors on thesecells.

In one embodiment of any method, cells, or composition described herein,the histone methyltransferase inhibited multipotent hematopoieticprogenitor cells of step (b) is co-culture with a stromal cell thatexpress a Notch ligand.

In one embodiment of any method, cells, or composition described herein,the co-culturing of cells occurs in a medium comprising Flt-3L and IL-7.

In one embodiment of any method, cells, or composition described herein,the co-culturing of cells is performed in serum-free culture conditions.

In one embodiment of any method, cells, or composition described herein,the cell expressing the Notch ligand is an OP-9 cell. In one embodiment,the OP-9 cell expresses DLL1, otherwise referred to as OP9-DL1 cells. Inanother embodiment, the OP-9 cell expresses DLL4, otherwise referred toas OP9-DL4 cells.

In one embodiment of any method, cells, or composition described herein,the notch ligand is Delta-like-1 (DLL1), Delta-like-4 (DLL4), andimmobolized Delta1^(ext-IgG), consisting of the extracellular domain ofhuman Delta-like-1 (DLL1) fused to the Fc domain of human IgG1.“Immobolized Delta1ext-IgG” refers to recombinant Notch ligand made byfusing the extracellular domain of Delta-like 1 to the Fc domain ofhuman IgG1. This is a synthetic way of providing a titratable dose ofNOTCH ligand. Varnum-Finney B et al. Immobilization of Notch ligand,Delta-1, is required for induction of notch signaling. J Cell Sci. 2000,23:4313-8. These references are incorporated herein by reference intheir entirety. Recombinant Notch ligands and Fc-fusions arecommercially available at AdipoGen™.

In one embodiment of any method, cells, or composition described herein,the DLL1 or DLL4 is supplied with co-culturing the multilineagehematopoietic progenitor cells with immobolized Delta1^(ext-IgG),OP9-DL1 cells or OP9-DL4 cells. OP9-DL1 cells are a bone-marrow-derivedstromal cell line that ectopically expresses the Notch ligand,Delta-like 1 (DLL1).

In one embodiment of any method, cells, or composition described herein,the Notch ligand is DLL1 or DLL4.

Method of differentiating progenitor cells to T-cells using the Notchsignaling pathway and OP9-Notch ligand expressing cells are known in theart. Any method can be used herein to produce the engineered immune fromthe multilineage hematopoietic progenitor cells that had previously beeninhibited with a histone methyltransferase inhibitor. For examples, asdescribed in the Example section and also as described in U.S. Pat. Nos.7,575,925, 8,772,028, 8,871,510, and 9,206,394 and US Patent PublicationNos: 20090217403, 20110123502, 20110052554 20110027881, 20110236363,20120149100, 20130281304, 20140322808, 20140248248, and 20140037599.These references are incorporated herein by reference in their entirety.

For differentiation in the lymphoid lineage and B cell lineagecommitment in the histone methyltransferase inhibited multipotenthematopoietic progenitor cells, these cells are exposed to (1) a B-cellpriming factors; (2) co-culturing with supporting cells expressing oneor more B-lineage growth factors; (3) co-culturing with supporting cellsexpressing CD40L in the absence or presence of one or more B-cellactivators; (4) exposure to one or more B-cell activators; or acombination of (1)-(4) over period of time in culture.

In some embodiments, a B-cell priming factor can also be a B-lineagegrowth factor. In some embodiments, a B-lineage growth factor can alsobe a B-cell priming factor.

B-cell priming factors are known in the art. For examples, IL-3, Flt3ligand, thrombopoietin, stem cell factor (SCF), granulocytecolony-stimulating factor (G-CSF), granulocyte colony-stimulating factor(GM-CSF), IL-7, and IL-11. As used herein, the term “B-cell primingfactor” refers to any compounds that are capable of supporting orpromoting the commitment of hematopoietic stem cells and/or lymphoidprogenitor cells to B-lineage development. A compound can be a smallmolecule, a polypeptide, a protein, or a nucleic acid. Various B-cellpriming factors can be used in the methods and systems described herein.Examples of B-cell priming factors include, but are not limited to,interleukin 3 (IL-3), Flt3 ligand, thrombopoietin, stem cell factor(SCF), granulocyte colony-stimulating factor (G-CSF), granulocytemacrophage colony-stimulating factor (GM-CSF), interleukin 7 (IL-7),interleukin 11 (IL-11), anti-phosphatase (Sbfl), and mechano growthfactor (MGF). One of skill in the art will be able to select the amountof a B-cell priming factor to use based on the particular circumstances.Generally, from about 1 to about 1000 ng/ml of a B-cell priming factorcan be used in the methods or systems described herein; however, in thetypical situation from about 1 to about 100 ng/ml of a B-cell primingfactor can be used. However, in some situations, more or less amount ofa B-cell priming factor may be used. In situations where more than oneB-cell priming factor is used, the amount of each B-cell priming factormay the same, or the amount of each B-cell priming factor may bedifferent from each other.

B-cell activators are known in the art. For examples, CpG DNA, IL-2,IL-10, IL-15, IL-6, IFNα, and anti-CD40L. As used herein, the term“B-cell activator” refers to any compounds that are capable of promotingthe activation of naïve B cells, preferably the antigen-independentactivation of naïve B cells. B-cell activators can be small molecules,polypeptides, proteins or nucleic acids. Conventional methods can beused to determine if a compound has the ability of stimulatingantigen-independent activation of naïve B cell. For example, thecompound can be tested for the activation of naïve B cells isolated fromhuman peripheral blood. Non-limiting examples of B-cell activatorsinclude CpG DNA; cytokines, such as IL-2, IL-3, IL-4, IL-6, IL-10,IL-15, IFNα; anti-CD40L; and lactic acid. One of skill in the art willbe able to select the amount of a B-cell activator based on theparticular circumstances. Generally, from about 1 to about 1000 ng/ml ofa cytokine B-cell activator can be used in the methods or systemsdescribed herein; however, in the typical situation from about 1 toabout 150 ng/ml, or about 1 to about 100 ng/ml of a cytokine B-cellactivator can be used. However, in some situations, more or less amountmay be used. Generally, from about 0.1 to about 5 μM CpG DNA can beused; however, in the typical situation from about 0.5 to about 4 μM, orabout 1 to about 3.5 μM, or about 1.5 to about 3 μM, or about 2 to about2.5 μM CpG DNA can be used. In situations where more than one B-cellactivator is used, the amount of each B-cell activator may the same, orthe amount of each B-cell activator may be different from each other.

B-lineage growth factors are known in the art. For examples, pre-pro-Bcell growth-stimulating factor (PPBSF), insulin-like growth factor-1(IGF-1), interleukin 3 (IL-3), Flt3 ligand, thrombopoietin, stem cellfactor (SCF), granulocyte colony-stimulating factor (G-CSF), Granulocytemacrophage colony-stimulating factor (GM-CSF), interleukin 11 (IL-11),anti-phosphatase (Sbfl), and mechano growth factor (MGF). As usedherein, the term “B-lineage growth factor” refers to any compounds thatare capable of promoting one or more stages of B cell differentiationduring B-lineage development. B-lineage growth factors can be smallmolecules, polypeptides, proteins, or nucleic acids. Non-limitingexamples of the stages in B-lineage development include: the stage fromprogenitor B cells to early pro-B cells, the stage from early pro-Bcells to late pro-B cells, the stage from late pro-B cells to largepre-B cells, the stage from large pre-B cells to small pre-B cells, thestage from small pre-B cells to immature B cells, and the stage fromimmature B cells to mature B cells. Examples of B-lineage growth factorinclude, but are not limited to, interleukin 7 (IL-7), pre-pro-B cellgrowth-stimulating factor (PPBSF), insulin-like growth factor-1 (IGF-1),interleukin 3 (IL-3), Flt3 ligand, thrombopoietin, stem cell factor(SCF), granulocyte colony-stimulating factor (G-CSF), granulocytemacrophage colony-stimulating factor (GM-CSF), interleukin 11 (IL-11),anti-phosphatase (Sbfl), and mechano growth factor (MGF). One of skillin the art will be able to select the amount of a B-lineage growthfactor based on the particular circumstances. Generally, from about 1 toabout 1000 ng/ml of a B-lineage growth factor can be used in the methodsor systems described herein. However, in the typical situation fromabout 1 to about 300 ng/ml, about 20 to about 200 ng/ml, about 50 toabout 150 ng/ml, about 80 to about 150 ng/ml of a cytokine B-cellactivator can be used. However, in some situations, more or less amountof a B-lineage growth factor may be used. In situations where more thanone B-lineage growth factor is used, the amount of each B-lineage growthfactor may the same, or the amount of each B-lineage growth factor maybe different from each other.

Supporting cells used in co-cultures for cell differentiation purposesare typically stromal cells. Various stromal cells can be used in themethods described herein. Examples of stromal cell lines include, butare not limited to murine MS5 stromal cell line; murine bonemarrow-derived stromal cell lines, such as S10, S17, OP9 and BMS2 celllines; human marrow stromal cell lines such as those described in U.S.Pat. No. 5,879,940. This reference is incorporated herein by referencein its entirety. The supporting cell or stromal cell expresses one ormore B-lineage growth factors, for example, growth factors IL-7.

As used herein, the term “supporting cell or stromal cell” when used inthe context of cell differentiation refers to any cells that are capableof creating, promoting, or supporting a microenvironment for the growth,proliferation, differentiation, or expansion of multipotenthematopoietic progenitor cells or T cells or B cells. Suitablesupporting cells that can be used in the systems and methods disclosedherein include, but are not limited to, stromal cells and fibroblastcells.

In some embodiments, the histone methyltransferase inhibited multipotenthematopoietic progenitor cells are co-cultured with a population offirst supporting cells expressing one or more B-lineage growth factors.In an embodiment, the first supporting cells can express IL-7. Inanother embodiment, the first supporting cells can express IL-7 and atleast one B-lineage growth factor selected from pre-pro-B cellgrowth-stimulating factor (PPBSF), insulin-like growth factor-1 (IGF-1),interleukin 3 (IL-3), Flt3 ligand, thrombopoietin, stem cell factor(SCF), granulocyte colony-stimulating factor (G-CSF), Granulocytemacrophage colony-stimulating factor (GM-CSF), interleukin 11 (IL-11),anti-phosphatase (Sbfl), and mechano growth factor (MGF). In someembodiments, one or more B-lineage growth factors are from humans. Insome embodiments, all B-lineage growth factors are from humans. In someembodiments, one or more B-lineage growth factors are from mammals otherthan humans. In some embodiments, all B-lineage growth factors are frommammals other than humans.

Entry and commitment to the B cell lineage can be monitored by theappearance of B cell specific markers. Many early B-lineage markers areknown in the art. For instance, pro-B cells can be identified by CD19and CD10 co-expression (CD19+CD10+) and the lack of for expression ofsurrogate light chains.

Methods of differentiating progenitor cells to B-cells are known in theart. Any method can be used herein to produce the engineered immune fromthe multilineage hematopoietic progenitor cells that had previously beeninhibited with a histone methyltransferase inhibitor. For examples, U.S.Pat. Nos. 8,034,613, 8,133,727, and 8,206,979, and US Patent PublicationNos: 20030152558, 20040029271, 20050153443, 20100047854, 2012004036,20120040362, and 20140273211. These references are incorporated hereinby reference in their entirety.

Induced Pluripotent Stem Cells

In some embodiments, the pluripotent stem cells (PSCs) described hereinare derived from isolated induced pluripotent stem cells (iPSCs). Anadvantage of using iPSCs is that the cells can be derived from the samesubject to which the eventual immune cells would be reintroduced. Thatis, a somatic cell can be obtained from a subject, reprogrammed to aninduced pluripotent stem cell, and then transfected and differentiatedinto a modified immune cell to be administered to the subject (e.g.,autologous cells). Since the progenitors are essentially derived from anautologous source, the risk of engraftment rejection or allergicresponses is reduced compared to the use of cells from another subjector group of subjects. In some embodiments, the cells for generatingiPSCs are derived from non-autologous sources. In addition, the use ofiPSCs negates the need for cells obtained from an embryonic source.Thus, in one embodiment, the PSCs used in the disclosed methods are notembryonic stem cells.

Although differentiation is generally irreversible under physiologicalcontexts, several methods have been recently developed to reprogramsomatic cells to induced pluripotent stem cells. Exemplary methods areknown to those of skill in the art and are described briefly hereinbelow.

As used herein, the term “reprogramming” refers to a process that altersor reverses the differentiation state of a differentiated cell (e.g., asomatic cell). Stated another way, reprogramming refers to a process ofdriving the differentiation of a cell backwards to a moreundifferentiated or more primitive type of cell. It should be noted thatplacing many primary cells in culture can lead to some loss of fullydifferentiated characteristics. Thus, simply culturing such cellsincluded in the term differentiated cells does not render these cellsnon-differentiated cells (e.g., undifferentiated cells) or pluripotentcells. The transition of a differentiated cell to pluripotency requiresa reprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture. Reprogrammed cells also have thecharacteristic of the capacity of extended passaging without loss ofgrowth potential, relative to primary cell parents, which generally havecapacity for only a limited number of divisions in culture.

The cell to be reprogrammed can be either partially or terminallydifferentiated prior to reprogramming. In some embodiments,reprogramming encompasses complete reversion of the differentiationstate of a differentiated cell (e.g., a somatic cell) to a pluripotentstate or a multipotent state. In some embodiments, reprogrammingencompasses complete or partial reversion of the differentiation stateof a differentiated cell (e.g., a somatic cell) to an undifferentiatedcell (e.g., an embryonic-like cell). Reprogramming can result inexpression of particular genes by the cells, the expression of whichfurther contributes to reprogramming. In certain embodiments describedherein, reprogramming of a differentiated cell (e.g., a somatic cell)causes the differentiated cell to assume an undifferentiated state(e.g., is an undifferentiated cell). The resulting cells are referred toas “reprogrammed cells,” or “induced pluripotent stem cells (iPSCs oriPS cells).”

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a common myeloid stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods described herein can also be ofuse for such purposes, in some embodiments.

The specific approach or method used to generate pluripotent stem cellsfrom somatic cells (broadly referred to as “reprogramming”) is notcritical to the claimed invention. Thus, any method that re-programs asomatic cell to the pluripotent phenotype would be appropriate for usein the methods described herein.

Reprogramming methodologies for generating pluripotent cells usingdefined combinations of transcription factors have been described toinduce pluripotent stem cells from somatic cells. Yamanaka and Takahashiconverted mouse somatic cells to ES cell-like cells with expandeddevelopmental potential by the direct transduction of Oct4, Sox2, Klf4,and optionally c-Myc. See U.S. Pat. Nos. 8,058,065 and 9,045,738 toYamanaka and Takahashi. iPSCs resemble ES cells as they restore thepluripotency-associated transcriptional circuitry and much of theepigenetic landscape. In addition, mouse iPSCs satisfy all the standardassays for pluripotency: specifically, in vitro differentiation intocell types of the three germ layers, teratoma formation, contribution tochimeras, germline transmission, and tetraploid complementation.

Subsequent studies have shown that human iPS cells can be obtained usingsimilar transduction methods, and the transcription factor trio, OCT4,SOX2, and NANOG, has been established as the core set of transcriptionfactors that govern pluripotency. The production of iPS cells can beachieved by the introduction of nucleic acid sequences encoding stemcell-associated genes into an adult, somatic cell, using viral vectors.

iPS cells can be generated or derived from terminally differentiatedsomatic cells, as well as from adult stem cells, or somatic stem cells.That is, a non-pluripotent progenitor cell can be rendered pluripotentor multipotent by reprogramming. In such instances, it may not benecessary to include as many reprogramming factors as required toreprogram a terminally differentiated cell. Further, reprogramming canbe induced by the non-viral introduction of reprogramming factors, e.g.,by introducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 2010 Nov. 5; 7(5):618-30, this referenceis incorporated herein by reference in its entirety.). Reprogramming canbe achieved by introducing a combination of nucleic acids encoding stemcell-associated genes including, for example Oct-4 (also known asOct-3/4 or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28. In oneembodiment, reprogramming using the methods and compositions describedherein can further comprise introducing one or more of Oct-3/4, a memberof the Sox family, a member of the Klf family, and a member of the Mycfamily to a somatic cell. In one embodiment, the methods andcompositions described herein further comprise introducing one or moreof each of Oct 4, Sox2, Nanog, c-MYC and Klf4 for reprogramming. Asnoted above, the exact method used for reprogramming is not necessarilycritical to the methods and compositions described herein. However,where cells differentiated from the reprogrammed cells are to be usedin, e.g., human therapy, in one embodiment the reprogramming is noteffected by a method that alters the genome. Thus, in such embodiments,reprogramming is achieved, e.g., without the use of viral or plasmidvectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various small molecules as shown by Shi, Y., et al (2008)Cell-Stem Cell 2:525-528, Huangfu, D., et al (2008) Nature Biotechnology26(7):795-797, and Marson, A., et al (2008) Cell-Stem Cell 3:132-135.This reference is incorporated herein by reference in its entirety.Thus, an agent or combination of agents that enhance the efficiency orrate of induced pluripotent stem cell production can be used in theproduction of patient-specific or disease-specific iPSCs. Somenon-limiting examples of agents that enhance reprogramming efficiencyinclude soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histonemethyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferaseinhibitors, histone deacetylase (HDAC) inhibitors, valproic acid,5′-azacytidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA),vitamin C, and trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HCToxin, Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VPA)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994(e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g.,6-(3-chlorophenylureido)caproic hydroxamic acid), AOE(2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Otherreprogramming enhancing agents include, for example, dominant negativeforms of the HDACs (e.g., catalytically inactive forms), siRNAinhibitors of the HDACs, and antibodies that specifically bind to theHDACs. Such inhibitors are available, e.g., from BIOMOL International,Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, AtonPharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, andSigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers can be selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto,Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, a cellthat expresses Oct4 or Nanog is identified as pluripotent. Methods fordetecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses. In someembodiments, detection does not involve only RT-PCR, but also includesdetection of protein markers. Intracellular markers may be bestidentified via RT-PCR, while cell surface markers are readilyidentified, e.g., by immunocytochemistry.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate to cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells are introduced to nude mice and histologyand/or immunohistochemistry is performed on a tumor arising from thecells. The growth of a tumor comprising cells from all three germlayers, for example, further indicates that the cells are pluripotentstem cells.

Many US Patents and Patent Application Publications teach and describemethods of generating iPSCs and related subject matter. For examples,U.S. Pat. Nos. 9,347,044, 9,347,042, 9,347,045, 9,340,775, 9,341,625,9,340,772, 9,250,230, 9,132,152, 9,045,738, 9,005,975, 9,005,976,8,927,277, 8,993,329, 8,900,871, 8,852,941, 8,802,438, 8,691,574,8,735,150, 8,765,470, 8,058,065, 8,048,675, and US Patent PublicationNos: 20090227032, 20100210014, 20110250692, 20110201110, 20110200568,20110306516, 20100021437, 20110256626, 20110044961, 20120276070,20120263689, 20120128655, 20120100568, 20130295064, 20130029866,20130189786, 20130295579, 20130130387, 20130157365, 20140234973,20140227736, 20140093486, 20140301988, 20140170746, 20140178989,20140349401, 20140065227, and 20150140662. These references areincorporated herein by reference in their entirety.

Somatic Cells for Reprogramming

Somatic cells, as that term is used herein, refer to any cells formingthe body of an organism, excluding germline cells. Every cell type inthe mammalian body—apart from the sperm and ova, the cells from whichthey are made (gametocytes) and undifferentiated stem cells—is adifferentiated somatic cell. For example, internal organs, skin, bones,blood, and connective tissue are all made up of differentiated somaticcells.

Additional somatic cell types for use with the compositions and methodsdescribed herein include: a fibroblast (e.g., a primary fibroblast), amuscle cell (e.g., a myocyte), a cumulus cell, a neural cell, a mammarycell, an hepatocyte and a pancreatic islet cell. In some embodiments,the somatic cell is a primary cell line or is the progeny of a primaryor secondary cell line. In some embodiments, the somatic cell isobtained from a human sample, e.g., a hair follicle, a blood sample, abiopsy (e.g., a skin biopsy or an adipose biopsy), a swab sample (e.g.,an oral swab sample), and is thus a human somatic cell.

Some non-limiting examples of differentiated somatic cells include, butare not limited to, epithelial, endothelial, neuronal, adipose, cardiac,skeletal muscle, skin, immune cells, hepatic, splenic, lung, peripheralcirculating blood cells, gastrointestinal, renal, bone marrow, andpancreatic cells. In some embodiments, a somatic cell can be a primarycell isolated from any somatic tissue including, but not limited tobrain, liver, gut, stomach, intestine, fat, muscle, uterus, skin,spleen, endocrine organ, bone, etc. Further, the somatic cell can befrom any mammalian species, with non-limiting examples including amurine, bovine, simian, porcine, equine, ovine, or human cell. In someembodiments, the somatic cell is a human somatic cell.

When reprogrammed cells are used for generation of thyroid progenitorcells to be used in the therapeutic treatment of disease, it isdesirable, but not required, to use somatic cells isolated from thepatient being treated. For example, somatic cells involved in diseases,and somatic cells participating in therapeutic treatment of diseases andthe like can be used. In some embodiments, a method for selecting thereprogrammed cells from a heterogeneous population comprisingreprogrammed cells and somatic cells they were derived or generated fromcan be performed by any known means. For example, a drug resistance geneor the like, such as a selectable marker gene can be used to isolate thereprogrammed cells using the selectable marker as an index.

Reprogrammed somatic cells as disclosed herein can express any number ofpluripotent cell markers, including: alkaline phosphatase (AP); ABCG2;stage specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60;TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; β-III-tubulin; α-smoothmuscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1;zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cellassociated transcript 1 (ECAT1); ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7;ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sal14;undifferentiated embryonic cell transcription factor (Utf1); Rex1; p53;G3PDH; telomerase, including TERT; silent X chromosome genes; Dnmt3a;Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog/ECAT4;Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3;CYP25A1; developmental pluripotency-associated 2 (DPPA2); T-celllymphoma breakpoint 1 (Tcl1); DPPA3/Stella; DPPA4; other general markersfor pluripotency, etc. Other markers can include Dnmt3L; Sox15; Stat3;Grb2; β-catenin, and Bmi1. Such cells can also be characterized by thedown-regulation of markers characteristic of the somatic cell from whichthe induced pluripotent stem cell is derived.

Uses of the Engineered Immune Cells Derived from Pluripotent Stem Cells

In one embodiment, provided herein a population of engineered immunecells produced by a method described herein, where in the cell comprisesan exogenous gene coding copy of each of the transcription factors: ERG,HOXA9, and RORA, and optionally, further comprising an exogenous genecoding copy of each of the transcription factors: SOX4, and MYB, orfurther comprising an exogenous gene coding copy of each of thetranscription factors: DACH1 and NFIA, or further comprising anexogenous gene coding copy of each of the transcription factors: SOX4,MYB, DACH1 and NFIA, In one embodiment, the population of cells furthercomprises a pharmaceutically acceptable carrier. These engineered immunecells can be culture expanded to increase the number of cells for use.

The engineered immune cells described herein are useful in thelaboratory for biological studies. For examples, these cells can bederived from an individual having a genetic disease or defect, and usedin the laboratory to study the biological aspects of the disease ordefect, and to screen and test for potential remedy for that disease ordefect.

Alternatively, the engineered immune cells described herein are usefulin cellular replacement therapy and other medical treatment in subjectshaving the need. For example, patients who have undergone chemotherapyor irradiation or both, and manifest deficiencies in immune functionand/or lymphocyte reconstitution, or in cancer immune therapy.

In various embodiments, the engineered immune cells described herein areadministered (ie., implanted or transplanted) to a subject in need ofcellular replacement therapy.

In one embodiment, provided herein is a method of cellular replacementtherapy, or for the treatment of cancer, autoimmune disorders,hematological diseases, or other genetic diseases and disorders in asubject, comprising (a) providing a somatic cell from a donor subject,(b) generating multilineage hematopoietic progenitor cells from myeloidprogenitor cells derived from the somatic cell as described in any ofthe preceding paragraphs; (c) inhibiting a histone methyltransferase inthe resultant population of multilineage hematopoietic progenitor cellsas described in any of the preceding paragraphs; (d) differentiating theresultant population of multilineage hematopoietic progenitor cells inthe presence of a notch ligand or a stromal cell or both to promotedifferentiation into the lymphoid lineage as described in any of thepreceding paragraphs, and (e) implanting or administering the resultantdifferentiated lymphoid cells into a recipient subject.

In one embodiment of the treatment method described above, the hostsubject and the recipient subject are the same individual.

In one embodiment of the treatment method described above, the hostsubject and the recipient subject are not the same individual, but areat least HLA compatible.

Hematologic diseases are disorders which primarily affect the blood.Non-limiting such diseases or disorders include myeloid deriveddisorders such as hemoglobinopathies (congenital abnormality of thehemoglobin molecule or of the rate of hemoglobin synthesis), examples,sickle-cell disease, thalassemia, and methemoglobinemia; Anemias (lackof red blood cells or hemoglobin), Pernicious anemia; disordersresulting in decreased numbers of cells, such as myelodysplasticsyndrome, neutropenia (decrease in the number of neutrophils), andthrombotic thrombocytopenic purpura (TTP), thrombocytosis, tematologicalmalignancies such as lymphomas, myelomas, and leukemia. Lymphomas suchas Hodgkin's disease, Non-Hodgkin's lymphoma, Burkitt's lymphoma,Anaplastic large cell lymphoma, Splenic marginal zone lymphoma,Hepatosplenic T-cell lymphoma, and Angioimmunoblastic T-cell lymphoma(AILT); myelomas such as Multiple myeloma, Waldenströmmacroglobulinemia, Plasmacytoma; leukemias that increases defect WBCsuch as Acute lymphocytic leukemia (ALL), Chronic lymphocytic leukemia(CLL), Acute myelogenous leukemia (AML), Chronic IdiopathicMyelofibrosis (MF), Chronic myelogenous leukemia (CML), T-cellprolymphocytic leukemia (T-PLL), B-cell prolymphocytic leukemia (B-PLL),Chronic neutrophilic leukemia (CNL), Hairy cell leukemia (HCL), T-celllarge granular lymphocyte leukemia (T-LGL), and Aggressive NK-cellleukemia.

Autoimmune diseases such as diabetes, rheumatoid arthritis and multiplesclerosis.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably in the context of the placementof described cells, e.g. hematopoietic progenitor cells, into a subject,by a method or route which results in at least partial localization ofthe introduced cells at a desired site, such as a site of injury orrepair, such that a desired effect(s) is produced. The cells e.g.hematopoietic progenitor cells, or their differentiated progeny can beadministered by any appropriate route which results in delivery to adesired location in the subject where at least a portion of theimplanted cells or components of the cells remain viable.

In various embodiments, the engineered immune cells described herein areoptionally expanded ex vivo prior to administration to a subject. Inother embodiments, the engineered immune cells are optionallycryopreserved for a period, then thawed prior to administration to asubject.

The engineered immune cells used for cellular replacement therapy can beautologous/autogeneic (“self”) or non-autologous (“non-self,” e.g.,allogeneic, syngeneic or xenogeneic) in relation to the recipient of thecells. “Autologous,” as used herein, refers to cells from the samesubject. “Allogeneic,” as used herein, refers to cells of the samespecies that differ genetically to the cell in comparison. “Syngeneic,”as used herein, refers to cells of a different subject that aregenetically identical to the cell in comparison. “Xenogeneic,” as usedherein, refers to cells of a different species to the cell incomparison. In preferred embodiments, the cells of the invention areallogeneic.

In various embodiments, the engineered immune cell described herein thatis to be implanted into a subject in need thereof is autologous orallogeneic to the subject.

In various embodiments, the engineered immune cell described herein canbe derived from one or more donors, or can be obtained from anautologous source. In some embodiments of the aspects described herein,the engineered immune cells are expanded in culture prior toadministration to a subject in need thereof.

In various embodiments, the engineered immune cell described herein canbe derived from one or more donors, or can be obtained from anautologous source.

In various embodiments, prior to implantation, the recipient subject istreated with chemotherapy and/or radiation.

In one embodiment, the chemotherapy and/or radiation is to reduceendogenous stem cells to facilitate engraftment of the implanted cells.

In various embodiments, prior to implantation, the engineered immunecells or the inhibited, reverse-lineage multilineage hematopoieticprogenitor cells are treated ex vivo with prostaglandin E2 and/orantioxidant N-acetyl-L-cysteine (NAC) to promote subsequent engraftmentin a recipient subject.

In various embodiments, the recipient subject is a human.

In various embodiments, the subject is diagnosed with HIV or other viraldisease, a hematological disease, or undergoing a cancer treatment.

In one aspect of any method, cells and composition described herein, asubject is selected to donate a somatic cell which would be used toproduce iPSCs and an engineered immune cell described herein. In oneembodiment, the selected subject has a genetic disease or defect.

In various embodiments, the donor subject is a human.

In various embodiments, the donor or the recipient subject is an animal,human or non-human, and rodent or non-rodent. For example, the subjectcan be any mammal, e.g., a human, other primate, pig, rodent such asmouse or rat, rabbit, guinea pig, hamster, cow, horse, cat, dog, sheepor goat, or a non-mammal such as a bird.

In various embodiments, the donor or the recipient subject is diagnosedwith HIV, a hematological disease or cancer.

In one aspect of any method, cells and composition described herein, abiological sample or a population of embryonic stem cells, somatic stemcells, progenitor cells, bone marrow cells, hematopoietic stem cells, orhematopoietic progenitor cells is obtained from the donor subject.

In various embodiments, biological sample or a population of embryonicstem cells, somatic stem cells, progenitor cells, bone marrow cells,hematopoietic stem cells, or hematopoietic progenitor cells describedherein can be derived from one or more donors, or can be obtained froman autologous source.

In one embodiment, the embryonic stem cells, somatic stem cells,progenitor cells, bone marrow cells, hematopoietic stem cells,hematopoietic progenitor cells are isolated from the donor subject,transfected, cultured (optional), and transplanted back into the samesubject, i. e. an autologous cell transplant. Here, the donor and therecipient subject is the same individual. In another embodiment, theembryonic stem cells, somatic stem cells, progenitor cells, bone marrowcells, hematopoietic stem cells, or hematopoietic progenitor cells areisolated from a donor who is an HLA-type match with a subject(recipient). Donor-recipient antigen type-matching is well known in theart. The HLA-types include HLA-A, HLA-B, HLA-C, and HLA-D. Theserepresent the minimum number of cell surface antigen matching requiredfor transplantation. That is the transfected cells are transplanted intoa different subject, i.e., allogeneic to the recipient host subject. Thedonor's or subject's embryonic stem cells, somatic stem cells,progenitor cells, bone marrow cells, hematopoietic stem cells, orhematopoietic progenitor cells can be transfected with a vector ornucleic acid comprising the nucleic acid molecule(s) described herein,the transfected cells are cultured, inhibited, and differentiated asdisclosed, optionally expanded, and then transplanted into the recipientsubject. In one embodiment, the transplanted engineered immune cellsengrafts in the recipient subject. In one embodiment, the transplantedengineered immune cells reconstitute the immune system in the recipientsubject. The transfected cells can also be cryopreserved aftertransfected and stored, or cryopreserved after cell expansion andstored.

The engineered immune cells or the histone methyltransferase inhibited,reverse-lineage multilineage hematopoietic progenitor cells may beadministered as part of a bone marrow or cord blood transplant in anindividual that has or has not undergone bone marrow ablative therapy.In one embodiment, genetically modified cells contemplated herein areadministered in a bone marrow transplant to an individual that hasundergone chemoablative or radioablative bone marrow therapy.

In one embodiment, a dose of cells is delivered to a subjectintravenously. In one embodiment, the cells are intravenouslyadministered to a subject.

In particular embodiments, patients receive a dose of the modified cellsdescribed herein, e.g., engineered immune cells or the histonemethyltransferase inhibited, reverse-lineage multilineage hematopoieticprogenitor cells, of about 1×10⁵ cells/kg, about 5×10⁵ cells/kg, about1×10⁶ cells/kg, about 2×10⁶ cells/kg, about 3×10⁶ cells/kg, about 4×10⁶cells/kg, about 5×10⁶ cells/kg, about 6×10⁶ cells/kg, about 7×10⁶cells/kg, about 8×10⁶ cells/kg, about 9×10⁶ cells/kg, about 1×10⁷cells/kg, about 5×10⁷ cells/kg, about 1×10⁸ cells/kg, or more in onesingle intravenous dose.

In certain embodiments, patients receive a dose of the modified cellsdescribed herein, e.g., engineered immune cells or the histonemethyltransferase inhibited, reverse-lineage multilineage hematopoieticprogenitor cells, of at least 1×10⁵ cells/kg, at least 5×10⁵ cells/kg,at least 1×10⁶ cells/kg, at least 2×10⁶ cells/kg, at least 3×10⁶cells/kg, at least 4×10⁶ cells/kg, at least 5×10⁶ cells/kg, at least6×10⁶ cells/kg, at least 7×10⁶ cells/kg, at least 8×10⁶ cells/kg, atleast 9×10⁶ cells/kg, at least 1×10⁷ cells/kg, at least 5×10⁷ cells/kg,at least 1×10⁸ cells/kg, or more in one single intravenous dose.

In an additional embodiment, patients receive a dose of the modifiedcells described herein, e.g., engineered immune cells or the histonemethyltransferase inhibited, reverse-lineage multilineage hematopoieticprogenitor cells, of about 1×10⁵ cells/kg to about 1×10⁸ cells/kg, about1×10⁶ cells/kg to about 1×10⁸ cells/kg, about 1×10⁶ cells/kg to about9×10⁶ cells/kg, about 2×10⁶ cells/kg to about 8×10⁶ cells/kg, about2×10⁶ cells/kg to about 8×10⁶ cells/kg, about 2×10⁶ cells/kg to about5×10⁶ cells/kg, about 3×10⁶ cells/kg to about 5×10⁶ cells/kg, about3×10⁶ cells/kg to about 4×10⁸ cells/kg, or any intervening dose ofcells/kg.

In general, the engineered immune cells or the histone methyltransferaseinhibited, reverse-lineage multilineage hematopoietic progenitor celldescribed herein are administered as a suspension with apharmaceutically acceptable carrier. For example, as therapeuticcompositions. Therapeutic compositions contain a physiologicallytolerable carrier together with the cell composition and optionally atleast one additional bioactive agent as described herein, dissolved ordispersed therein as an active ingredient. In a preferred embodiment,the therapeutic composition is not substantially immunogenic whenadministered to a mammal or human patient for therapeutic purposes,unless so desired. One of skill in the art will recognize that apharmaceutically acceptable carrier to be used in a cell compositionwill not include buffers, compounds, cryopreservation agents,preservatives, or other agents in amounts that substantially interferewith the viability of the cells to be delivered to the subject. Aformulation comprising cells can include e.g., osmotic buffers thatpermit cell membrane integrity to be maintained, and optionally,nutrients to maintain cell viability or enhance engraftment uponadministration. Such formulations and suspensions are known to those ofskill in the art and/or can be adapted for use with the cells asdescribed herein using routine experimentation.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike. A pharmaceutically acceptable carrier will not promote the raisingof an immune response to an agent with which it is admixed, unless sodesired. The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well understood inthe art and need not be limited based on formulation. Typically suchcompositions are prepared as injectable either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance theeffectiveness of the active ingredient. The therapeutic composition ofthe present invention can include pharmaceutically acceptable salts ofthe components therein. Pharmaceutically acceptable salts include theacid addition salts (formed with the free amino groups of thepolypeptide) that are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,tartaric, mandelic and the like. Salts formed with the free carboxylgroups can also be derived from inorganic bases such as, for example,sodium, potassium, ammonium, calcium or ferric hydroxides, and suchorganic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine and the like. Physiologically tolerable carriers arewell known in the art. Exemplary liquid carriers are sterile aqueoussolutions that contain no materials in addition to the activeingredients and water, or contain a buffer such as sodium phosphate atphysiological pH value, physiological saline or both, such asphosphate-buffered saline. Still further, aqueous carriers can containmore than one buffer salt, as well as salts such as sodium and potassiumchlorides, dextrose, polyethylene glycol and other solutes. Liquidcompositions can also contain liquid phases in addition to and to theexclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions. The amount of an active agent used in the methods describedherein that will be effective in the treatment of a particular disorderor condition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. Suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences, A. Osol, a standard reference text in this field of art. Forexample, a parenteral composition suitable for administration byinjection is prepared by dissolving 1.5% by weight of active ingredientin 0.9% sodium chloride solution.

In one embodiment, the “pharmaceutically acceptable” carrier does notinclude in vitro cell culture media.

In some embodiments, the composition of engineered immune cellsdescribed further comprises a pharmaceutically acceptable carrier. Inone embodiment, the pharmaceutically acceptable carrier does not includetissue or cell culture media.

In various embodiments, a second or subsequent dose of cells isadministered to the recipient subject. For example, second andsubsequent administrations can be given between about one day to 30weeks from the previous administration. Two, three, four or more totaladministrations can be delivered to the individual, as needed.

A cell composition can be administered by any appropriate route whichresults in effective cellular replacement treatment in the subject, i.e.administration results in delivery to a desired location in the subjectwhere at least a portion of the composition delivered, i.e. at least1×10⁴ cells are delivered to the desired site for a period of time.Modes of administration include injection, infusion, or instillation,“Injection” includes, without limitation, intravenous, intra-arterial,intraventricular, intracardiac injection and infusion. For the deliveryof cells, administration by injection or infusion is generallypreferred.

Efficacy testing can be performed during the course of treatment usingthe methods described herein. Measurements of the degree of severity ofa number of symptoms associated with a particular ailment are notedprior to the start of a treatment and then at later specific time periodafter the start of the treatment.

The present invention can be defined in any of the following numberedparagraphs:

-   -   [1]. A method comprising:        -   a. generating multilineage hematopoietic progenitor cells            from myeloid progenitor cells;        -   b. inhibiting a histone methyltransferase in the resultant            population of multilineage hematopoietic progenitor cells;            and        -   c. differentiating the resultant population of multilineage            hematopoietic progenitor cells in the presence of a notch            ligand or a stromal cell or both to promote differentiation            into the lymphoid lineage.    -   [2]. A method comprising:        -   a. in vitro transfecting myeloid progenitor cells with an            exogenous gene coding copy of each of the following            transcription factors ERG, HOXA9, and RORA, wherein the            transcription factors are expressed in the transfected cells            to produce a population of multilineage hematopoietic            progenitor cells that having myeloid and erythroid            potential;        -   b. inhibiting a histone methyltransferase in the resultant            population of multilineage hematopoietic progenitor cells to            expand lymphoid potential; and        -   c. differentiating the resultant population of multilineage            hematopoietic progenitor cells in the presence of a notch            ligand or supportive stroma or both to promote            differentiation into the lymphoid lineage.    -   [3]. The method of paragraph 1, wherein the multilineage        hematopoietic progenitor cells are produced by introducing in        vitro each of the following transcription factors ERG, HOXA9,        RORA, in the myeloid progenitor cells.    -   [4]. The method of paragraph 2 or 3, further comprising        transfecting the myeloid progenitor cells with an exogenous gene        coding copy of the transcription factor, SOX4, and MYB.    -   [5]. The method of paragraph 2, 3, or 4, further comprising        transfecting the myeloid progenitor cells with an exogenous gene        coding copy of the transcription factor, NFIA and DACH1.    -   [6]. The method of any one of paragraphs 1-5, wherein the        myeloid lineage progenitor cells are CD34+CD45+.    -   [7]. The method of any one of paragraphs 1-6, wherein the        multilineage hematopoietic progenitor cells are CD34+CD38        negative/low.    -   [8]. The method of any one of paragraphs 1-7, wherein the        myeloid lineage progenitor cells are embryoid body progenitor        cells derived from a population of pluripotent stem cells.    -   [9]. The method of paragraph 8, wherein the population of        pluripotent stem cells is induced pluripotent stem cells (iPS        cells) or embryonic stem cells (ESC).    -   [10]. The method of paragraph 9, wherein the induced pluripotent        stem cells are produced by introducing only reprogramming        factors OCT4, SOX2, KLF4 and optionally c-MYC or nanog and LIN28        into mature cells.    -   [11]. The method of paragraph 10, wherein the mature cells are        selected from the group consisting of B lymphocytes (B-cells), T        lymphocytes, (T-cells), fibroblasts, and keratinocytes.    -   [12]. The method of paragraph 9, 10 or 11, wherein the induced        pluripotent stem cells are produced by introducing the        reprogramming factors two or more times into the mature cells.    -   [13]. The method of any one of paragraphs 1-12, wherein the        notch ligand is selected from the group consisting of        Delta-like-1, Delta-like-4, and immobolized Delta1^(ext-IgG),        which consisting of the extracellular domain of human        Delta-like-1 fused to the Fc domain of human IgG1.    -   [14]. The method of paragraph 13, wherein the Delta-like-1 or        Delta-like-4 is supplied with co-culturing the multilineage        hematopoietic progenitor cells with immobolized        Delta1^(ext-IgG), OP9-DL1 cells or OP9-DL4 cells.    -   [15]. The method of any one of paragraphs 1-14, wherein the        histone methyltransferase catalyses the addition of methyl group        to the histone H3 lysine residue 9 (H3K9) and/or histone H3        lysine residue 27 (H3K27).    -   [16]. The method of paragraph 15, wherein the histone        methyltransferase H3K9 and/or H3K27 is inhibited by a small        molecule or a nucleic acid.    -   [17]. The method of paragraph 16, wherein the histone        methyltransferase H3K9 and/or H3K27 small molecule inhibitor is        an organic or inorganic compound having a molecular weight of        less than about 10,000 grams per mole or a salt, or ester or        other pharmaceutically acceptable form of said compound, a        peptide, a peptidomimetic, an amino acid, an amino acid analog,        a nucleotide, or a nucleotide analog.    -   [18]. The method of paragraph 16 or 17, wherein the histone        methyltransferase H3K9 and/or H3K27 small molecule inhibitor is        a heterorganic compound or an organometallic compound.    -   [19]. The method of any one of claims 16-18, wherein the small        molecule inhibitor is selected from the group consisting of        BIX-01294, UNC0638, E72, BRD4770, A-366, chaetocin, UNC0224,        UNC0631, UNC0646, EPZ005687, EPZ-6438 (E7438), 3-deazaneplanocin        A (DZNep), EI1, GSK343, GSK126, and UNC1999.    -   [20]. The method of paragraph 16, wherein the nucleic acid        inhibitor is a nucleic acid targeting the expression of histone        methyltransferase.    -   [21]. The method of paragraph 16 or 17, wherein the nucleic acid        inhibitor is a RNA interference inhibitor or agent.    -   [22]. The method of paragraph 21, wherein the nucleic acid        inhibitor is a EZH1 specific nucleic acid that is selected from        the group consisting of an aptamer that binds EZH1, a EZH1        specific RNA interference agent, or a vector encoding a EZH1        specific RNA interference agent, wherein the RNA interference        agent comprises one or more of the nucleotide sequences selected        from the group consisting of SEQ ID NO: 1-5, 27-30.    -   [23]. An immune cell produced by a method of any one of        paragraphs 1-22.    -   [24]. An immune cell derived from a population of myeloid        progenitor cells, wherein the immune cell comprises an exogenous        copy of each of the following transcription factors ERG, HOXA9,        and RORA.    -   [25]. The immune cell of paragraph 24, wherein the immune cell        further comprises an exogenous copy of each of the following        reprogramming factors SOX4, and MYB    -   [26]. The immune cell of paragraph 24 or 25, wherein the immune        cell further comprises an exogenous copy of each of the        following reprogramming factors NFIA and DACH1.    -   [27]. The immune cell of paragraph 24, 25 or 26, wherein the        immune cell further comprises an exogenous copy of each of the        following reprogramming factors OCT4, SOX2, KLF4 and optionally        c-MYC.    -   [28]. The immune cell of any one of paragraphs 24-27, wherein        the cell is further genetically modified remove of the native T        cell receptor (TCR) locus, to deletion of class I or class II        major histocompatibility complexes or both, to express of        non-canonical HLA-G or HLA-E or both, or to edit endogenous HLA        therein.    -   [29]. A composition comprising a population of immune cells of        any one of paragraphs 23-28.    -   [30]. The composition of paragraph 29, further comprising a        pharmaceutically acceptable carrier.    -   [31]. A pharmaceutical composition comprising a population of        immune cells of any one of paragraphs 23-28 and a        pharmaceutically acceptable carrier.    -   [32]. A pharmaceutical composition of paragraph 31 for use in        cellular replacement therapy in a subject.    -   [33]. An ex vivo or in vitro method of improving in vivo        engraftment of hematopoietic cells in a host comprising:        -   a. generating multilineage hematopoietic progenitor cells            from myeloid progenitor cells according to the method            paragraphs 2-12;        -   b. inhibiting a histone methyltransferase in the resultant            population of multilineage hematopoietic progenitor cells            according to the method paragraphs 15-22;        -   c. differentiating the resultant population of multilineage            hematopoietic progenitor cells in the presence of a notch            ligand or supportive stroma or both to promote            differentiation into the lymphoid lineage according to            paragraphs 13-14, and        -   d. transplanting said resultant multilineage hematopoietic            progenitor cells into a host.    -   [34]. A method of cellular replacement therapy, or immunotherapy        in a subject in need thereof, the method comprising        administering a population of immune cells of paragraphs 23-28,        or a composition of paragraph 29-30, or a pharmaceutical        composition of paragraphs 31-32 to a recipient subject.    -   [35]. The method of cellular replacement therapy of paragraph        34, wherein the subject is a patient who has undergone        chemotherapy or irradiation or both, and manifest deficiencies        in immune function or lymphocyte reconstitution or both        deficiencies in immune function and lymphocyte reconstitution.    -   [36]. The method of cellular replacement therapy of paragraph 34        or 35, wherein the subject prior to implantation, the immune        cells are treated ex vivo with prostaglandin E2 and/or        antioxidant N-acetyl-L-cysteine (NAC) to promote subsequent        engraftment in a recipient subject.    -   [37]. The method of cellular replacement therapy of paragraph 34        or 35, wherein the immune cells are autologous to the recipient        subject or at least HLA type matched with the recipient subject.    -   [38]. A modified or an engineered myeloid progenitor cell having        reversed lineage that include increased lymphoid lineage        potential.    -   [39]. A composition comprising modified or engineered myeloid        progenitor cell having reversed lineage that include increased        lymphoid lineage potential.    -   [40]. A modified myeloid progenitor cell or a composition        comprising modified or engineered myeloid progenitor cell, the        modified myeloid progenitor cell having reversed lineage and has        increased lymphoid lineage potential, for use in the        manufacture/production of described modified immune cells,        wherein the modified myeloid progenitor cell comprises an        exogenous gene coding copy of each of the following        transcription factors: ERG, HOXA9, and RORA.    -   [41]. A modified myeloid progenitor cell or a composition        comprising modified or engineered myeloid progenitor cell, the        modified myeloid progenitor cell having reversed lineage and has        increased lymphoid lineage potential, for use in cellular        replacement therapy, or for the treatment of cancer, autoimmune        disorders, hematological diseases, or other genetic diseases and        disorders, wherein the modified myeloid progenitor cell        comprises an exogenous gene coding copy of each of the following        transcription factors: ERG, HOXA9, and RORA.    -   [42]. The modified myeloid progenitor cell of paragraphs 38-41        further comprises an exogenous gene coding copy of SOX4, or MYB,        or both SOX4 and MYB.    -   [43]. The modified myeloid progenitor cell of paragraphs 38-42        further comprises an exogenous gene coding copy of DACH1, or        NFIA, or both DACH1 and NFIA.    -   [44]. The modified myeloid progenitor cell of paragraphs 38-43        are derived from lineage-restricted CD34⁺CD45⁺ myeloid precursor        cells.    -   [45]. The modified myeloid progenitor cell of paragraphs 38-44        further comprises an exogenous copy of each of the following        reprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC.    -   [46]. A method of cellular replacement therapy, or for the        treatment of cancer, autoimmune disorders, hematological        diseases, or other genetic diseases and disorders in a subject,        comprising (a) providing a somatic cell from a donor        subject, (b) generating multilineage hematopoietic progenitor        cells from myeloid progenitor cells derived from the somatic        cell as described in any of the preceding paragraphs; (c)        inhibiting a histone methyltransferase in the resultant        population of multilineage hematopoietic progenitor cells as        described in any of the preceding paragraphs; (d)        differentiating the resultant population of multilineage        hematopoietic progenitor cells in the presence of a notch ligand        or a stromal cell or both to promote differentiation into the        lymphoid lineage as described in any of the preceding        paragraphs, and implanting the resultant differentiated lymphoid        cells into a recipient subject.    -   [47]. The method of paragraph 46, wherein the host subject and        the recipient subject are the same individual.    -   [48]. The method of paragraph 46, wherein the host subject and        the recipient subject are not the same individual, but are at        least HLA compatible.

This invention is further illustrated by the following example whichshould not be construed as limiting. The contents of all referencescited throughout this application, as well as the figures and table areincorporated herein by reference.

Those skilled in the art will recognize, or be able to ascertain usingnot more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Example Experimental Procedures

hIPSC culture. All experiments were performed using MSC-IPS1 (Park etal., 2008), CD34-IPS and CD45-IPS. Human IPS cells were maintained onmouse embryonic fibroblasts (GlobalStem) feeders in DMEM/F12+20%KnockOut-Serum Replacement (Invitrogen™), 1 mM L-glutamine, 1 mM NEAA,0.1 mM β-mercaptoethanol, and 10 ng/ml bFGF. Media was changed daily,and cells were passaged 1:4 onto fresh feeders every 7 days usingstandard clump passaging with collagenase IV.

EB differentiation. EB differentiation was performed as previouslydescribed (Chadwick et al., 2003). Briefly, hPSC colonies were scrapedinto non-adherent rotating 10 cm plates at the ratio of 2:1. The EBmedia was KO-DMEM+20% FBS (Stem Cell Technologies), 1 mM L-glutamine, 1mM NEAA, penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 200 μg/mlh-transferrin, and 50 μg/ml ascorbic acid. After 24 hrs, media waschanged by allowing EBs to settle by gravity, and replaced with EB mediasupplemented with growth factors: 50 ng/ml BMP4 (R&D Systems), 200 ng/mlSCF, 200 ng/ml FLT3, 50 ng/ml G-CSF, 20 ng/ml IL-6, 10 ng/ml IL-3 (allPeprotech). Media was changed on day 5, and day 10. EBs were dissociatedon day 14 by digesting with collagenase B (Roche) for 2 hrs, followed bytreatment with enzyme-free dissociation buffer (Gibco), and filteredthrough an 80 μm filter. Dissociated EBs were frozen in 10% DMSO, 40%FBS freezing solution.

Progenitor sorting. Dissociated EB cells were thawed following the LonzaPoietics protocol which can be found at the website of Lonza, under thesection of manuals and instructions for the procedure for thawingpoietics cells. The thawed cells are resuspended at 1×10⁶ per 100 μlstaining buffer (PBS+2% FBS). CD34+ cells were sorted from bulk EBculture using human CD34 microbeads (Miltenyi Biotec) and run through amagnetic column separator (MACS) as per manufacturer's instructions.

Lentiviral and shRNA library plasmids. 5F lentiviral plasmids: HOXA9,ERG, RORA, SOX4, and MYB were cloned into pInducer-21 Dox-induciblelentiviral vector. Four shRNAs for each epigenetic modifier and threeshRNAs for luciferase were obtained from the Broad Institute RNAiConsortium in pLKO.1 or pLKO.5 lentiviral vectors. Lenti-viral particleswere produced by transfecting 293T-17 cells (ATCC) with the lentiviralplasmids and 3rd-generation packaging plasmids. Virus was harvested 24hours after transfection and concentrated by ultracentrifugation at23,000 rpm for 3 hrs. All viruses were titered by serial dilution on293T cells. All TRC numbers for shRNAs used in this study are providedin Table 1.

5F gene transfer and 5F culture. MACS separated CD34⁺ EB progenitorswere seeded on retronectin-coated (10 μg/cm²) 96 well plates at adensity of 2-5×10⁴ cells per well. The infection media was SFEM(StemCell) with 50 ng/ml SCF, 50 ng/ml FLT3, 50 ng/ml TPO, 50 ng/ml IL6,10 ng/ml IL3 (all R&D Systems). Lentiviral infections were carried outin a total volume of 150 μl. The multiplicity of infection (MOI) for thefactors was MOI=5 for ERG and HOXA9, MOI=3 for RORA, SOX4, MYB, andMOI=2 for all shRNAs. Virus was concentrated onto cells by centrifugingthe plate at 2300 rpm for 30 min at RT. Infections were carried out for24 hrs. After gene transfer, 5F cells were cultured in SFEM with 50ng/ml SCF, 50 ng/ml FLT3, 50 ng/ml TPO, 50 ng/ml IL6, and 10 ng/ml IL3(all R&D Systems). Dox was added at 2 μg/ml (Sigma). Cultures weremaintained at a density of <1×10⁶ cells/ml, and media were changed every3-4 days. After 14 days of culture, 5F were plated in the T celldifferentiation protocol.

Co-cultures with Notch ligand delta like 1-expressing OP9 (0P9-DL1) celllines. The OP9-DL1 cells were cultured as monolayers in OP9 media, whichis α-MEM supplemented with FCS (20% final conc.), 2-mercaptoethanol (0.1mM final conc.), nonessential amino acids (0.1 mM final conc,), sodiumpyruvate (1 mM final conc.), penicillin (10 U/ml final conc.),L-glutamine (1 mM final conc.), streptomycin (100 μg/ml final conc.),and sodium bicarbonate (2.2 g/liter final conc.).

Reverse lineage, multipotent hematopoietic progenitor cells (also knownas the EB-derived progenitors described above) were plated on themonolayers of OP9-DL1 at a density of 1-6×10⁵ cells per well of a 6-wellor 100-mm non-treated dish. The culture media contained SCF (30 ng/mlfinal conc.; R&D systems), Flt3 ligand and IL-7 (5 ng/ml final conc.each; R&D systems). On day 7 of culture, loosely adherent hematopoieticcells were harvested by gentle pipetting. Every 5 days thereafter,non-adherent iPS cell-derived hematopoietic cells were collected byvigorous pipetting, filtered through a 70-μm nylon mesh, and transferredonto OP9-DL1 monolayers in OP9 media. All cytokines were added at allsubsequent passages.

By day 14 of co-culture with OP9-DL1 cells, the reverse lineage,multipotent hematopoietic progenitor cells were transformed intolymphocyte-like cells. These cells expressed CD25 and/or CD44 by day 14of coculture, and are considered to have been differentiated into Tlineage in the same way that progenitor cells differentiate in thethymus. Phycoerythrin-conjugated anti-CD8 antibody (clone 53-6.7),anti-CD19 antibody (clone 1D3) and anti-CD25 antibody (clone 7D4), andallophycocyanin-conjugated anti-CD4 antibody (clone GK1.5), anti-CD11bantibody (clone M1/70) and anti-CD44 antibody (clone IM7) (all fromBiolegend (Tokyo)) were used to verify expression of the CD cell surfacemarkers.

Rearrangement at the TCRβ locus (Tcrb) is a hallmark of T cell lineagecommitment and is essential for the progression of CD4/CD8 doublenegative thymocytes to the double positive stage during normal αβ T celldevelopment. To determine whether the T cells that develop from reverselineage, multipotent hematopoietic progenitor cells cultured on OP9-DL1cells undergo normal rearrangement of the TCRβ locus, the differentiatedcells were stained at day 30 with various antibodies against TCRβ chain.Fluorescein isothiocynate-conjugated TCR panel (BD biosciences) wasused.

It is expected that a diverse patterns of TcrVβ gene expression bedetected in the differentiated reverse lineage, multipotenthematopoietic progenitor cells. The diversity can be confirmed bygenomic PCR. OP9-DL1 cells and mouse adult thymocytes can be used aspositive controls for the genomic PCR-based analysis.Previously-reported PCR primers are used for the analysis of Tcr generearrangement (Ikawa T, Kawamoto H, Wright L Y et al., “Long-termcultured E2A-deficient hematopoietic progenitor cells are pluripotent”,Immunity, Vol. 20, pp. 349-360.; Kawamoto H, Ohmura K, Fujimoto S etal., “Extensive proliferation of T cell lineage-restricted progenitorsin the thymus: an essential process for clonal expression of diverse Tcell receptor beta chains” Eur. J. Immunol., Vol. 33, pp. 606-615.).

During normal thymocyte development, T cells bearing TCRαβ or TCRγδdevelop in the thymus. To determine whether both populations of T cellsdevelop from reverse lineage, multipotent hematopoietic progenitor cellscultured on OP9-DL1 cells, the differentiated reverse lineage,multipotent hematopoietic progenitor cells were analyzed for surfaceexpression of TCRαβ or TCRγδ using allophycocyanin-conjugated anti-TCRβantibody (clone H57-597), and phycoerythrin-conjugated anti-TCRγδantibody (clone GL3). It is expected that both αβ T cells and γδ T cellswere generated from differentiated cells in this coculture system.

Similarly, after day 20 co-culture, the differentiated cells are CD4/CD8double positive cells and CD8 single positive cells.

Confirmation of functional TCRs expressed on the co-culturedifferentiated cells. The CD4+CD8+ differentiated cells in thiscoculture system were sorted from the cultures at day 21, and 7.5×10⁴ Tcells were stimulated for 3 days with plate-bound anti-CD3 antibody (10μg/ml final conc.; clone 145-2C11) in the differentiation medium in thepresence of IL-2 (1 ng/ml final conc.) and anti-CD28 antibody (1 μg/mlfinal conc.; clone 37.51). After that, PMA/Ionomycin was added to theculture and the cell were exposed to the added PMA/Ionomycin for afurther 6 hour. Intracellular staining for IFN-γ was done withCytofix/Cytoperm® and GolgiStop® (BD Biosciences) according to themanufacturer's instructions. Phycoerythrin-conjugated anti-CD8 antibody(clone 53-6.7) and phycoerythrin-conjugated anti-IFN-γ antibody (cloneXMG1.2) were used for IFN-γ production analysis. The stained cells wereanalyzed by flow cytometry. It is expected that the CD4−CD8+differentiated cells in this coculture system would produce IFN-γ inresponse to the TCR stimulation by anti-CD3 antibody and anti-CD28antibody, and this indicate functional TCRs are expressed in thedifferentiated cells from reverse lineage, multipotent hematopoieticprogenitor cells.

Additionally, the 7.5×10⁴ of the CD4+CD8+ differentiated cells in thisco-culture system were cultured for 2 days with plate-bound anti-CD3antibody (10 μg/ml final conc.; clone 145-2C11) in differentiationmedium in the presence of IL-2 (2 ng/ml final conc.) and TGF-β1 (5 ng/mlfinal conc.). It is expected that there will be enhanced the populationof Foxp3-positive cells, which is the hallmark of regulatory T cells, asobserved in naïve T cells derived from normal adult lymphoid tissue(Chen W, Jin W, Hardegen N et al., “Conversion of peripheral CD4+CD25−naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction oftranscription factor Foxp3”, J. Exp. Med., Vol. 198, pp. 1875-1886.).These data indicate that the iPS cell-derived T cells generated in thiscoculture can respond to stimulation via TCR or cytokine receptors.

T cell differentiation. After 14 days of respecification, 1×10⁵ 5F wereplated in OP9-DL1 stromal co-culture. Cells were cultured in α-MEM(Gibco), 1% penicillin/streptomycin, 20% FBS (Gemini), 1 mM L-glutaminewith 30 ng/mL SCF, 5 ng/mL FLT3, 5 ng/mL (all R&D Systems) for 20 dayswith 2 ug/mL Dox followed by Dox removal. Cells were harvested bymechanical dissociation and filtered through a 40 uM cap and passagedonto fresh stroma every 5-7 days. T cell development was assessed after35 days using CD45, CD7, CD3, CD4 and CD8.

Mouse YS or AGM cells were dissociated to single cells for 30 minutes inEB dissociation media containing 250 mg Collagenase IV, 100 mgHyaluronidase V, 6.8 mg DNase I in 50 mL DMEM (10×) diluted to 1× withIMDM. Cells across multiple embryos were pooled and counted. 75K cellswere seeded onto one confluent well of OP9-DL1 in a 6-well plate. Cellswere cultured in aMEM, 20% serum, 1% Pen/Strep/L-glutamine, 5 ng/mLmIL-7 (R&D) and 5 ng/mL hFLT3 (R&D) for 12 days.

B cell differentiation. After 14 days of respecification, 5×10⁴ 5F wereplated into a single well of MS-5 stroma in a 6-well plate. Cells werecultured in Myelocult H5100 (Stem Cell Technologies) supplemented with1% penicillin/streptomycin 50 ng/mL SCF (R&D), 10 ng/mL FLT3 (R&D), 25ng/mL IL7 (R&D) and 25 ng/mL TPO (R&D) for 10 days with 2 ug/mL Doxfollowed by Dox removal.

For murine B cell differentiation, YS or EP pooled from multiple embryosof the same genotype were dissociated to single cells and 75K cells wereseeded onto a confluent well of OP9 stroma in a 6-well plate. Cells werecultured in aMEM, 10% serum, 5×10⁻⁵ M B-mercaptoethanol, 1% PSG, 50 U/mLmIL7 (R&D) and 10 ng/mL hFLT3 (R&D).

For the class-switching assay, B cell progenitors were purified usingthe B220 MACS or CD19 MACS microbead enrichment kit (Miltenyi Biotec) asper manufacturer's recommendations. 5×10⁵ B220⁺ or 5×10⁵ CD19⁺ cellswere plated into one well of a 6-well plate in complete RPMI mediasupplemented with 0.5 ug/mL anti-CD40 (Ebioscience, Cat. #16-0402-86)and 25 ng/mL IL4 (Ebioscience, Cat. #14-8041). Class-switching wasanalyzed by flow on day 4.

Colony assays. After 14 days of respecification, 5×10⁴ cells were platedinto 3 ml of complete methylcellulose H3434 (StemCell Technologies)supplemented with 10 ng/ml IL6 (Peprotech), 10 ng/ml FLT3 (R&D), and 50ng/ml TPO (R&D). The mixture was distributed into two 60 mm dishes andmaintained in a humidified chamber for 14 days.

Mouse transplantation. NOD/LtSz-scidIL2Rgnull (NSG) (Jackson Labs) micewere bred and housed at the Boston Children's Hospital animal carefacility. Animal experiments were performed in accordance toinstitutional guidelines approved by BCH animal care committee.Intravenous transplants have been previously described. Briefly, 6-10week old mice were irradiated (275 rads) 24 hrs before transplant. Toensure consistency between experiments, only female mice were used.Cells were transplanted in a 100 uL volume using a 28.5 g insulinneedle. Sulfatrim was administered in drinking water to preventinfections after irradiation.

Flow cytometry. The following antibodies were used for human cells: CD45APC-Cy7 (557833, BD Biosciences), CD4 PE-Cy5 (IM2636U, Beckman CoulterImmunotech), CD8 BV421 (RPA-T8, BD Horizon), CD5 BV510 (UCHT2, BDBiosciences), TCRgd APC (555718, BD Biosciences), TCRab BV510(T10B9.1A-31, BD Biosciences), CD3 PE-Cy7 (UCHT1, BD Pharmigen), CD7 PE(555361, BD Pharmigen), CD1a APC (559775, BD Pharmigen) for T cellstaining. For B cell staining: CD45 PE-Cy5 (IM2652U, Beckman CoulterImmunotech), CD19 PE (4G7, BD), CD56 V450 (B159, BD Biosciences), CD11bAPC-Cy7 (557754, BD Biosciences), For HSC/Progenitor sorting: CD34PE-Cy7 (8G12, BD), CD45 (557833, BD Biosciences), CD38 PE-Cy7 (IM2651U,BD), DAPI. For myeloid and erythroid staining: CD11b APC-Cy7 (557754, BDBiosciences), GLYA PE-Cy7 (A71564, Beckman Coulter), CD71 PE (555537, BDBiosciences), CD45 PE-Cy5 (IM2652U, Beckman Coulter Immunotech). Allstains were performed with <1×106 cells per 100 μl staining buffer(PBS+2% FBS) with 1:100 dilution of each antibody, 30 min at RT in dark.Compensation was performed by automated compensation with anti-mouse Igkand negative beads (BD). All acquisition was performed on BD Fortessa orBD Aria cytometer.

The following antibodies were used for mouse cells: CD45.2 PE-Cy7 (104,eBioscience), CD45.1 FITC (A20, eBioscience), B220 PB (RA3-6B2, BDBiosciences), Ter119 PE-Cy5 (Ter 119, eBioscience), GR1 (RB6-8C5, BDBioscience), CD3 APC (145-2C11, eBioscience), CD19 APC-Cy7 (1D3, BDBioscience), MAC1 A700 (M1/70, BD Bioscience) for engraftment analyses.For HSC/progenitor staining: CD45.2 APC-Cy7 (104, BioLegend), Sca1PE-Cy7 (D7, BD Bioscience), CD34 AF700 (RAM34, eBioscience), CD48 FITC(BioLegend), CD150 PE-Cy5 (TC15-12F12.2, BioLegend), cKit APC (2B8,eBioscience), Fcγ PE (93, eBioscience). For RNA seq sort: CD16/32 (93,Bio-legend), Ter119 Biotin (Ter119, eBioscience), GR1 Biotin (RB6-8C,eBioscience), CD3 Biotin (17A2, eBioscience), CD5 Biotin (53-6.7,eBioscience), CD19 Biotin (eBio1D3, eBioscience), Streptavidin EF450(eBioscience), CD45 PerCP-Cy5.5 (30-F11, eBioscience), CD144 EF660(eBioBV13, eBioscience), CD117 APC-EF780 (2B8, eBioscience), CD41 PE-Cy7(eBioMWReg30, eBioscience). For B cell staining: CD45.2 APC-CY7 (104,BioLegend), CD23 PE-Cy7 (B3B4, eBioscience), Ter119 PE-Cy5 (Ter 119,eBioscience), IgM EF660 (eB121-15F9, eBioscience), MAC1 A700 (M1/70, BDBioscience), CD5 BV510 (53-7.3 BD Biosciences), IgM (11/41,eBioscience), IgG1 FITC (A85-1, BD), B220 PE Cy5 (RA3-6B2, BDBiosciences). For T cell staining: CD45.2 APC-CY7 (104, BioLegend), TCRbPE-Cy5 (H57-597, BD Biosciences), CD8 APC-EF780 (53-6.7, eBioscience),CD4 APC (GK1.5, eBioscience), CD3 AF700 (17A2, BioLegend), TCRgd FITC(GL3, BD Biosciences). All stains were performed with <1×106 cells per100 μl staining buffer (PBS+2% FBS) with 1:100 dilution of eachantibody, 30 min on ice in dark. Compensation was performed by automatedcompensation with anti-mouse Igk and negative beads (BD). Allacquisition was performed on BD Fortessa or BD Aria cytometer.

Results

The inventors have previously demonstrated that it is possible torespecify primitive progenitors with limited lymphoid differentiationpotential. To expand the selection of candidate factors, the inventorsscreened epigenetic modifiers to provide an additional regulatory layerfor the respecification. The inventors employed a library of shorthairpin RNAs (shRNAs) from the Broad RNAi Consortium to target 20 genesin DNA and histone methylation pathways (FIG. 7A) previously used in thelab to enhance efficiency of reprogramming to pluripotency (Onder T. etal. 2012).

Using the established respecification platform, the inventorsdifferentiated two iPSC lines (MSC-IPS, CD45-IPS) into embryoid bodies(EB) under hematopoietic promoting conditions to generate CD34⁺CD45⁺myeloid progenitors. The inventors then transduced EB-derivedprogenitors with the five transcription factors (5F cocktail: ERG,HOXA9, RORA, SOX4 and MYB) and infected with individual shRNAs targetingeach epigenetic modifier and screened for T lymphoid potential using theestablished OP9-DL1 co-culture system. The results from threeindependent screens are summarized in FIGS. 1A-1F. After validation oftop hits, the lead candidate was EZH1.

EZH1 is a Critical Repressor of Definitive Potential.

To test the hypothesis that epigenetic factors act as barriers todefinitive potential, the inventors adopted a loss-of-functionphenotypic screen using an shRNA library targeting 20 DNA and histonemethylation factors previously shown to affect somatic cellreprogramming (FIG. 7A) (Onder et al. 2012). The inventors introducedthe library into primitive CD34⁺ progenitors derived from embryoid bodydifferentiation. To facilitate phenotypic screening, the inventorsexpanded these primitive progenitors using a defined set of 5transcription factors (5F) (Doulatov et al 2013). Expanded progenitorsretained embryonic features, including lack of B and T cell potential,and expression of embryonic globins. The inventors transduced 5F cellsthe individual hairpins (4x hairpins per gene), and screened for theemergence of T cell potential using the OP9-DL1 co-culture system(Holmes and Zúñiga-Pflücker 2009) (FIGS. 1A and 1F). After five weeks ofco-culture, T cell potential was analyzed by flow cytometry for CD7,CD3, CD4 and CD8. The inventors found that knockdown of eight epigeneticregulators enhanced CD4⁺CD8⁺ T cell potential from primitive 5F cells(FIG. 1B). Among the top hits were several members of the methylated DNAbinding proteins, histone H3 lysine 9 (H3K9) methyltransferases, PRC2components, and SMYD2, a SET domain-containing methyltransferase (FIG.1C). H3K9 and H3K27 methyltransferases have been previously linked tolineage commitment and self-renewal in fetal and adult HSCs (Ugarte etal. 2015, Chen et al. 2012, Xie et al. 2014, Hidalgo et al. 2012, Lee etal. 2015).

To prospectively validate these top candidates, the inventors analyzed Tcell potential of 5F cells transduced with shRNAs (2x per gene)targeting these 8 factors. Independently, the inventors tested their Blymphoid potential using co-culture with MS-5, a murine bonemarrow-derived stroma that supports B cell differentiation (Nishihara etal. 1998, Ohkawara et al. 1998). 5F cells transduced with multipleshRNAs targeting a control luciferase gene displayed none or negligible(interval, with standard deviations) levels of T and B cell potential.Of the tested candidates, only knockdown of EZH1 elicited robust T and Bcell potential across independent hairpins and iPSC lines (FIGS. 2A, 2B,2E). Myelo-erythroid differentiation potential of progenitors transducedwith shRNAs for EZH1 (5F+shEZH1) was largely unchanged as compared toshRNAs targeting luciferase (5F+shLUC), by flow cytometry (FIGS. 2C, 2D)and colony-forming assays (FIG. 2F). These findings indicate that lossof EZH1 uncovers multilymphoid potential in primitive hematopoieticprogenitors.

Other PRC2 Components do not Phenocopy EZH1.

EZH1 is a member of the Polycomb group proteins. Polycomb repressivecomplex 2 (PRC2) mediates methylation on histone H3 lysine 27 (H3K27)and plays critical roles in transcriptional regulation and stem celldevelopment. EZH1 and EZH2 are closely related enzymatic subunits ofPRC2. The well-characterized canonical PRC2 is comprised of EZH2, EEDand SUZ (Onder T. et al., 2012). EZH1 was identified as a homolog ofEZH2 and an interacting partner to EED (Jones C A et al. 1998 and ShenX. et al. 2008) and believed to play redundant or compensatory roles forEZH2. Recent work, however, has uncovered novel gene activating rolesfor EZH1 in addition to its transcriptional repression functions(Mousavi K. et al. 2012 and Xu J. et al. 2015).

EZH1 is a homolog of Drosophila Enhancer of zeste E(z) (Abel et al.1996), a catalytic component of PRC2 (Laible et al. 1997, Jones et al.1998, Shen et al. 2008). PRC2 is comprised of E(z), Eed and Suz12, whichmediate epigenetic silencing at developmentally regulated genes (Mulleret al. 2002, Sparmann and van Lohuizen 2006, Simon and Kingston 2009).While EZH2 is the primary catalytic component of PRC2, EZH1 canfunctionally substitute for Ezh2 (Shen et al. 2008), although Ezh1 has aweaker methyltransferase activity (Magueron et al. 2008) and can promoteRNA polymerase elongation (Mousavi et al. 2012). Accumulating evidenceindicates that EZH1 and EZH2 have distinct molecular functions. Ezh1,unlike Ezh2, is most frequently found by itself or in complex withnucleosome-recognizing protein Suz12. By contrast, Ezh2 is almost alwayscomplexed with Suz12 and scaffold protein Eed. In addition, while EZH2is required for somatic reprogramming, loss of EZH1 enhancesreprogramming (Onder et al. 2012, Cacchiarelli et al. 2015). Thesestudies illustrate a complex mode of epigenetic regulation by PRC2depending on subunit interactions and holoenzyme composition.

To dissect the importance of PRC2 in definitive hematopoietic potential,the inventors knocked down each component of PRC2 (2 shRNAs per gene) in5F cells and assessed T cell differentiation. The inventors confirmedefficient knockdown by each shRNA (FIG. 3A). EZH1 knockdown dramaticallyenhanced the percentage of CD4⁺CD8⁺ T cells (5-fold vs shLUC). Knockdownof SUZ12 also enhanced T cell potential, albeit to a lesser extent.However, knockdown of EED and EZH2 did not affect T cell potential(FIGS. 3A and 3C). EZH1 and EZH2 dual knockdown phenocopied EZH2depletion, indicating that EZH2 is epistatic to EZH1 (FIG. 3B). Thus,loss of EZH2 does not phenocopy EZH1 in restoring definitive potential.To validate this finding, the inventors used an EZH2/EZH1 dualinhibitor, GSK126, that has 150-fold higher selectivity for EZH2 overEZH1 (McCabe et al. 2012) (FIGS. 3G, 3I and 3J). At 3 uM of GSK126, theinventors observed markedly reduced global H3K27me3 with partialtoxicity, as assessed by colony-forming assays (FIGS. 3I, 3J). To testthe effect of EZH2 inhibition on T cell potential, the inventorsgenerated definitive hemogenic endothelium (HE) from hPSCs with Tlymphoid potential via inhibition of Activin/Nodal signaling at theearly stage of mesoderm differentiation (Kennedy et al. 2012). InDMSO-treated cells, the inventors observed a robust CD4+CD8+ T cellpopulation, which was abrogated upon treatment with 3 uM GSK126,indicating that EZH2 unlike EZH1 is required for T cell differentiation(FIG. 3H).

To determine whether the catalytic SET domain of EZH1 was required torestrict definitive hematopoietic potential, the inventors performedrescue experiments with the full-length murine Ezh1 open reading frame(ORF) (mEzh1) or Ezh1 with the catalytic SET domain deleted (mEzh1ASET)to escape targeting by shRNAs targeting human EZH1 (FIG. 3D). Theinventors observed no T cells in any condition with 5F+shLUC, and arobust population of CD4+CD8+ T cells in 5F+shEZH1, as before (FIG. 3E,3F). Co-expression of mEzh1 completely abrogated T cell potential of5F+shEZH1 cells (FIG. 3E, 3F), indicating that the murine ORF issufficient to functionally restrict definitive hematopoietic potential.By contrast, expression of mEzh1ASET did not repress T cell potential(FIG. 3E, 3F). Taken together, these data indicate that specific EZH1inhibition rather than general PRC2 inhibition unlocks definitivehematopoietic potential and the catalytic SET domain is required torestrict this potential.

EZH1 directly regulates HSC and lymphoid genes. To understand themolecular basis for enhanced definitive potential, the inventorsperformed RNA sequencing analysis of CD34⁺CD38⁻ 5F+shLUC and 5F+shEZH1cells. Genes significantly upregulated following EZH1 knockdown (104genes, >2-fold, t-test, p<0.1) were enriched for gene ontology (GO)terms defense response, immune response and T cell costimulation (FIGS.4A, 4B). To specifically analyze the transcriptional changes associatedwith the human HSPC hierarchy, the inventors performed GSEA using thesix signatures that capture earliest patterns of lineage commitment(Doulatov et al. 2010): HSC, MLP (early lymphoid), HSC_MLP (stem andlymphoid), GMP (myeloid), CMP_MEP (erythroid) and Progenitor (allprogenitors). HSC_MLP and MLP signatures were highly enriched in5F+shEZH1 (FIG. 4C), consistent with acquisition of lymphoid potential.

The inventors next performed ATAC-sequencing to identify differentialregions of chromatin accessibility (Buenrostro et al. 2013). UnbiasedGREAT analysis (McLean et al. 2010) of the 1500 ATAC peaks significantlyupregulated upon EZH1 knockdown revealed enrichment in pathways relatedto T cell development, lymphocyte activation and immune response (FIG.4E, FIG. 9A). Conversely, downregulated peaks were enriched in pathwaysrelated to other cell developmental processes such as re-productiveprocess, neural and lung development, and importantly embryonichematopoiesis (FIG. 4G, FIG. 9B). Furthermore, HSC, HSC/MLP, B and Tcell signatures were all significantly enriched among upregulated ATACpeaks (FIG. 4F, FIG. 9C), indicating that EZH1 knockdown inducesepigenetic remodeling to unlock accessibility to HSC andlymphoid-associated genes.

To determine if the changes in chromatin accessibility and geneexpression were directly induced by EZH1, the inventors defined itsgenome-wide occupancy by overexpressing an epitope-tagged EZH1 or EZH2in 5F cells followed by ChIP-sequencing. Comparison of EZH2 and EZH1binding sites at promoter regions identified 1069 unique EZH1 bindingsites (FIG. 5A) that were associated with repressive, bivalent andactive marks (FIGS. 5B, 5C). To better annotate these EZH1 bindingsites, the inventors defined the transcriptions factors (TF, 152 out of1069 genes) that were uniquely bound and compared them to the TFsignatures of early HSPC hierarchy (Laurenti et al. 2013). Strikingly,EZH1-bound TFs were highly enriched in HSC, MLP and Pro-B populations(FIGS. 5D and 5F), and a large number of these were bivalently marked(FIG. 5G). Of all the EZH1-bound bivalent genes, a significant number ofgenes are annotated as granulocyte/macrophage, NK-, T- and B-cellspecific genes (Novershtern et al. 2011) (FIG. 5H). Although EZH1-boundTFs did not show significant alterations in expression, the regulatednetworks controlled by each EZH1-bound TF defined by CellNet weresignificantly changed (FIG. 5D). Specifically, the EZH1-bound TFs andtheir networks were significantly enriched in the HSPC, B and T cellGRNs (FIG. 5E). The networks of EZH1-bound TFs such as STAT5A, YAP1,NLRC5, ZNF697 and BACH2 were all significantly upregulated in B and Tcell GRNs (Data not shown). Taken together, these data providecompelling evidence that EZH1 directly binds to HSC and MLPtranscription factors, and inhibition of EZH1 unlocks definitivehematopoietic potential by de-repressing stem and lymphoid generegulatory networks.

Ezh1 deficiency enhances embryonic lymphopoiesis in vivo. To interrogatethe role of Ezh1 in vivo, the inventors first investigated earlylymphoid development in murine embryos. The extra-embryonic yolk sac(YS) is the earliest site of hematopoiesis and was thought to generateonly primitive erythroid and myelo-erythroid progenitors (Dzierzak andSpeck 2008). However, recent studies have reported lymphoid potential inthe yolk sac before definitive HSC emergence (Boiers et al. 2013,Yoshimoto et al. 2012, Yoshimoto et al. 2011). To determine whether Ezh1deficiency can enhance this early lymphoid potential, the inventorsperformed lineage analysis of E9.5 yolk sac (YS) from wild-type (WT) andEzh1 knockout (Ezh1−/−) embryos. The inventors detected a smallpopulation of CD19⁺B220⁺ B cells (3.64%) as well as CD3⁺CD5⁺ T cells(0.45%) (FIGS. 11A-11C) in the WT YS. These lymphoid populations trendedtoward higher frequencies in Ezh1−/− YS, although this increase was notsignificant (FIGS. 11B and 11C). Furthermore, the inventors could notrule out the possibility of circulating maternal blood confounding ourlineage analysis. Therefore, the inventors dissected yolk sac away fromE9.5 WT and Ezh1^(−/−) embryo proper and performed in vitro lymphoiddifferentiation. The inventors detected a robust population of embryonicB cells (B-1 subtype; AA4.1⁺CD19⁺B220^(lo-neg)), and few adult-like B-2cells (AA4.1⁺CD19⁺B220⁺) consistent with previous findings (Yoshimoto etal. 2012) (FIG. 12A). The inventors did not find significant differencesin B cell potential between WT and Ezh1^(−/−) cells, but note anincrease in class-switching from germline IgM to IgG1 (FIG. 12C).Similarly, the inventors did not observe differences in T cell potentialbetween WT and Ezh1^(−/−) in the embryo proper (EP) (FIGS. 6A and 6B).By contrast, Ezh1^(−/−) YS progenitors generated 2-5-fold more CD4⁺CD8⁺T cells (FIGS. 6B and 6D). These early T cells predominantly expressedfetal TCRγδ, though a small proportion expressed adult-type TCRβ (FIGS.6C and 6D). These data demonstrate that Ezh1-deficiency enhanceslymphoid potential of YS progenitors. Taken together, the findings thusfar propose a role for EZH1 in repressing lymphoid lineage fate, as asurrogate measure of definitive potential.

Ezh1 haploinsufficiency promotes generation of HSCs in ontogeny. Theemergence of bona fide HSCs, defined by the capacity to repopulate adultrecipients, marks the transition from embryonic to definitivehematopoiesis. If EZH1 acts as a gatekeeper of definitive potential, theinventors predicted that HSCs may emerge earlier and dis-play enhancedrepopulating potential. While HSCs appear in the AGM around 10.5 dpc(Boisset et al. 2010), they are extremely rare (˜1 HSC/embryo) and donot robustly support engraftment of adult hosts (Bertrand et al. 2005,Muller et al. 1994, North et al. 2002). Thus, focusing on thistransitional time point, the inventors isolated AGM and YS from E10.5WT, Ezh1^(+/−) and Ezh1^(−/−) embryos (FIG. 6E). Expression of Ezh1 andSuz12 decreased from YS to AGM, while Ezh2 and Eed were higher in theAGM (FIG. 6F). The inventors transplanted whole AGM (3.5 embryoequivalents (ee)) or YS (5 ee) into sub-lethally irradiated adultNOD/SCID-IL2Rγ^(null) (NSG) mice and monitored hematopoieticreconstitution. Engraftment from WT AGM was observed in 3/7 mice(11.9±13.6%) after 4 weeks, but decreased over time, with 2/7 miceengrafted (12.2±11.4%) after 16 weeks (FIG. 6G). This corresponds to 1repopulating unit in −10.4 embryo equivalents (ee). Only 1/7 WTAGM-transplanted mice displayed long-term multi-lineage chimerism,consistent with HSCs being exceedingly rare at this time. By contrast,5/8 mice transplanted with Ezh1^(−/−) AGM-derived cells were engraftedafter 4 weeks (36.7±20.9%) and retained stable chimerism over time (16weeks; 34.3±32.9%). Even more notably, mice transplanted with Ezh1+/−cells had the highest initial chimerism (41.2±29.2%; 4/5 engrafted),which increased over time (68.9±35.6%), and was predominantlymulti-lineage (3/5 mice). (FIGS. 6G and 6H). This corresponds to 1repopulating unit in 3.6 Ezh1−/− and 2.2 Ezh1+/− embryo equivalents, anearly 5-fold increase in frequency of HSCs.

Similarly to the AGM, YS at E10.5 contains few if any HSCs. Consistentwith this, we detected low level engraftment of WT YS cells in 5/7recipients after 4 weeks (3.4±1.5%), but only 3/9 mice after 16 weeks(4.3±2.9%) (FIG. 6I). By contrast, most Ezh1−/− (6.0±4.9%, 5/7engrafted), and all of Ezh1+/−YS-transplanted mice (8.8±6.5%, 5/5engrafted), showed stable long-term engraftment (FIGS. 61 and 6M). Thenumber of repopulating units was similar to the AGM (˜1 in 8.9 ee WT; 1in 4 Ezh1−/−, 1 in <2 Ezh1+/−). All engrafted mice were reconstitutedwith myeloid and lymphoid lineages (FIG. 6J). The inventors observed asignificant increase in the T cell graft of Ezh1−/− AGM transplantrecipients compared with WT AGM recipients (FIG. 6J). Up to 80% of Bcells in the peritoneal cavity of Ezh1+/− AGM-engrafted mice were of theadult-like B-2, as opposed to the embryonic B-1 cells (FIG. 13A).Furthermore, >90% of donor-derived CD45.2+CD3+ T cells expressedadult-type TCRβ, as opposed to embryonic TCRγδ configuration, in Ezh1−/−and Ezh1+/− AGM and YS engrafted mice (FIG. 13B). These data providecompelling evidence that Ezh1 deficiency, and especiallyhaploinsufficiency, stimulates generation of definitive HSCs andadult-like lymphopoiesis.

To determine the extended self-renewal potential of Ezh1-deficient HSCs,the inventors performed secondary transplantation. Using 1% as thecutoff for engraftment, the inventors did not detect any donorcontribution in mice transplanted with WT AGM (0/4 mice) or YS (0/7mice) in the peripheral blood after 4 weeks. By contrast, 4/7 Ezh1−/−(4.4±1.0%) and 9/9 Ezh1+/−(57.8±30.6%) AGM-transplanted secondaryrecipients were engrafted. (FIGS. 6K and 6L). While no Ezh1^(−/−) YSmice (0/10) were engrafted, the inventors observed chimerism fromEzh1^(+/−) YS cells (5/7 mice engrafted, 1.5±0.7%), which increased by16 weeks post-transplantation (6/7 mice engrafted, 5.3±4.5%) (FIGS. 6Mand 6N). Notably, all of the secondary recipients of Ezh1-deficient AGMand YS displayed multi-lineage engraftment with B, T, and myeloidlineages. (FIGS. 6L and 6N) Taken together, Ezh1 uniquely repressesdefinitive potential during ontogeny, and Ezh1-deficiency promoteslong-term, multilineage differentiation and self-renewal potential ofembryonic stem and progenitor cells.

The references cited herein and throughout the specification areincorporated herein by reference.

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TABLE 1Candidate target transcription factors for RNA interference and the corresponding shRNAPreferred SEQ. NCBI Taxon Gene ID. Clone ID Symbol Gene ID ID NameRegion Target Seq NO.: TRCN0000355903 NM_024670.3 SUV39H2 79723 96063UTR TAATGGAAGGCAGACTATTTA  31 TRCN0000355904 NM_024670.3 SUV39H2 797239606 CDS CTCTAATGACAAGCATAATTA  32 TRCN0000355905 NM_024670.3 SUV39H279723 9606 3UTR TCAAGGTTCTACCTATGTTAA  33 TRCN0000011057 NM_024670.3SUV39H2 79723 9606 CDS GCCCACCTTCAGACTTCTATT  34 TRCN0000275322NM_003173.2 SUV39H1  6839 9606 CDS GTACGTGGGAGAGATCATTAC  35TRCN0000275321 NM_003173.2 SUV39H1  6839 9606 CDS GACTGAGTCCTGCCGCAAATA 36 TRCN0000275372 NM_003173.2 SUV39H1  6839 9606 CDSAGTCGAGTACCTGTGCGATTA  37 TRCN0000158337 NM_003173.1 SUV39H1  6839 96063UTR CGTTGGGATTCATGGCCTATT  38 TRCN0000040076 NM_004456.3 EZH2  21469606 CDS CGGAAATCTTAAACCAAGAAT  39 TRCN0000010475 NM_004456.3 EZH2  21469606 3UTR GAAACAGCTGCCTTAGCTTCA  40 TRCN0000018365 NM_004456.3 EZH2 2146 9606 CDS TATGATGGTTAACGGTGATCA  41 TRCN0000040073 NM_004456.3 EZH2 2146 9606 3UTR TATTGCCTTCTCACCAGCTGC  42 TRCN0000276085 NM_020197.2SMYD2 56950 9606 CDS CGGCAAAGATCATCCATATAT  43 TRCN0000276083NM_020197.2 SMYD2 56950 9606 CDS GCTGTGAAGGAGTTTGAATCA  44TRCN0000276154 NM_020197.2 SMYD2 56950 9606 CDS GCTCTGTGTTTGAGGACAGTA 45 TRCN0000276155 NM_020197.2 SMYD2 56950 9606 3UTRACTTAGTTCAGAAACCTTAAA  46 TRCN0000036056 NM_024757.3 EHMT1 79813 9606CDS GCAACGGATACATCTTAAATA  47 TRCN0000036057 NM_024757.3 EHMT1 798139606 CDS CCTCGGTTCTGAGTCGTATAA  48 TRCN0000217965 NM_024757.3 EHMT179813 9606 CDS TCGAGAAGCTAGAGATCATAA  49 TRCN0000218919 NM_024757.3EHMT1 79813 9606 CDS ACCTCTTTGATCTCGACAATA  50 TRCN0000115668NM_025256.4 EHMT2 10919 9606 CDS CCTCTTCGACTTAGACAACAA  51TRCN0000115667 NM_025256.4 EHMT2 10919 9606 3UTR CACACATTCCTGACCAGAGAT 52 TRCN0000115670 NM_025256.4 EHMT2 10919 9606 CDSCGAGAGAGTTCATGGCTCTTT  53 TRCN0000115669 NM_025256.4 EHMT2 10919 9606CDS GCTCCAGGAATTTAACAAGAT  54 TRCN0000148112 NM_012432.2 SETDB1  98699606 CDS GCTCAGATGATAACTTCTGTA  55 TRCN0000072261 promegaLuc.1LUCIFERASE   -14 CONTROL CDS CACTCGGATATTTGATATGTG  56 TRCN0000276105NM_012432.2 SETDB1  9869 9606 CDS AGTTAGAGACATGGGTAATAC  57TRCN0000276106 NM_012432.2 SETDB1  9869 9606 CDS CGTGACTTCATAGAGGAGTAT 58 TRCN0000276103 NM_012432.2 SETDB1  9869 9606 3UTRATCCCTCCCATCCCATATTTG  59 TRCN0000297828 NM_003927.3 MBD2  8932 9606 CDSGTAGCAATGATGAGACCCTTT  60 TRCN0000013319 NM_003927.3 MBD2  8932 9606 CDSGCCTAGTAAATTACAGAAGAA  61 TRCN0000297830 NM_003927.3 MBD2  8932 9606 CDSGTACGCAAGAAATTGGAAGAA  62 TRCN0000013322 NM_003927.3 MBD2  8932 9606 CDSCTTGAATACAACATTGCCAAT  63 TRCN0000358468 NM_002384.2 MBDI  4152 96063UTR GCCCTTCCTCACAGAGTTAAA  64 TRCN0000072256 promegaLuc.1 LUCIFERASE  -14 CONTROL CDS ACGCTGAGTACTTCGAAATGT  65 TRCN0000358382 NM_002384.2MBD1  4152 9606 CDS GATGATTCTGCCTCCAAATTG  66 TRCN0000015429 NM_002384.1MBD1  4152 9606 CDS CCGGGAACAGAGAATGTTTAA  67 TRCN0000329862 NM_002384.2MBD1  4152 9606 CDS CACCCGTGATCACGGAGATTT  68 TRCN0000355735 NM_001991.3EZH1  2145 9606 CDS CTATCTGGCAGTGCGAGAATG   1 TRCN0000355734 NM_001991.3EZH1  2145 9606 CDS AGACGTGCAAGCAGGTCTTTC   2 TRCN0000378151 NM_001991.3EZH1  2145 9606 3UTR TGGATGACTTATGCGTGATTT   3 TRCN0000002442NM_001991.2 EZH1  2145 9606 CDS CAACAGAACTTTATGGTAGAA   4 TRCN0000021208NM_003797.2 EED  8726 9606 CDS CCAGTGAATCTAATGTGACTA  69 TRCN0000021205NM_003797.2 EED  8726 9606 CDS CCAGAGACATACATAGGAATT  70 TRCN0000381067NM_003797.2 EED  8726 9606 CDS GTGCGATGGTTAGGCGATTTG  71 TRCN0000021204NM_003797.2 EED  8726 9606 CDS GCAAACTTTATGTTTGGGATT  72 TRCN0000280721NM_002931.3 RING1  6015 9606 CDS CTGGAGCTGGTGAATGAGAAA  73TRCN0000021989 NM_002931.2 RING1  6015 9606 CDS GCCCTGATCTCTAAGATCTAT 74 TRCN0000280798 NM_002931.3 RING1  6015 9606 CDSGTCAGATCAGACCACAACGAT  75 TRCN0000352834 NM_002931.3 RING1  6015 9606CDS AGACGAGGTATGTGAAGACAA  76 TRCN0000229416 NM_005180.5 BMI1   648 9606CDS ATTGATGCCACAACCATAATA  77 TRCN0000218869 NM_005180.5 BMI1   648 9606CDS CAGATTGGATCGGAAAGTAAA  78 TRCN0000020156 NM_005180.5 BMI1   648 9606CDS CCTAATACTTTCCAGATTGAT  79 TRCN0000229418 NM_005180.5 BMI1   648 9606CDS TAATGGATATTGCCTACATTT  80 TRCN0000274442 NM_003926.5 MBD3 53615 9606CDS CAAGATGCTGATGAGCAAGAT  81 TRCN0000285209 NM_003926.5 MBD3 53615 9606CDS CGGCCTGAACGCCTTCGACAT  82 TRCN0000358524 NM_003926.5 MBD3 53615 9606CDS GACCTGAGCACCTTCGACTTC  83 TRCN0000274441 NM_003926.5 MBD3 53615 9606CDS GCCGGTGACCAAGATTACCAA  84 TRCN0000298921 NM_015355.2 SUZ12 235129606 CDS GCTGACAATCAAATGAATCAT  85 TRCN0000331118 NM_015355.2 SUZ1223512 9606 CDS CGGAATCTCATAGCACCAATA  86 TRCN0000038725 NM_015355.1SUZ12 23512 9606 CDS GCTTACGTTTACTGGTTTCTT  87 TRCN0000038726NM_015355.1 SUZ12 23512 9606 CDS CCAAACCTCTTGCCACTAGAA  88TRCN0000342689 NM_003925.1 MBD4  8930 9606 3UTR GCCTAGTGTGTGTGCTTTCTT 89 TRCN0000342754 NM_003925.1 MBD4  8930 9606 CDS GCAACGACTCTTACCGAATTT 90 TRCN0000342688 NM_003925.1 MBD4  8930 9606 CDS CCCACGACGTAAAGCCTTTAA 91 TRCN0000342752 NM_003925.1 MBD4  8930 9606 CDS GCCAAGTAGTAGTTCAGAGTT 92 TRCN0000021891 NM_001379.1 DNMT1  1786 9606 CDSGCCCAATGAGACTGACATCAA  93 TRCN0000072250 promegaLuc.1 LUCIFERASE   -14CONTROL CDS AGAATCGTCGTATGCAGTGAA  94 TRCN0000021893 NM_001379.1 DNMT1 1786 9606 CDS CGACTACATCAAAGGCAGCAA  95 TRCN0000232751 NM_001379.1DNMT1  1786 9606 3UTR GAGGTTCGCTTATCAACTAAT  96 TRCN0000232748NM_001379.1 DNMT1  1786 9606 CDS CCCGAGTATGCGCCCATATTT  97TRCN0000236345 NM_032482.2 DOT1L 84444 9606 CDS TCGCCAACACGAGTGTTATAT 98 TRCN0000020210 NM_032482.1 DOT1L 84444 9606 CDSCCGCAAGAAGAAGCTAAACAA  99 TRCN0000236342 NM_032482.2 DOT1L 84444 9606CDS CACATTGGAGAGAGGCGATTT 100 TRCN0000020211 NM_032482.1 DOT1L 844449606 CDS CCCGGATCTCAAGCTCGCTAT 101 TRCN0000035757 NM_022552.3 DNMT3A 1788 9606 CDS CCAGATGTTCTTCGCTAATAA 102 TRCN0000035756 NM_022552.3DNMT3A  1788 9606 CDS GCCTCAGAGCTATTACCCAAT 103 TRCN0000035754NM_022552.3 DNMT3A  1788 9606 CDS CCCAAGGTCAAGGAGATTATT 104TRCN0000035758 NM_022552.3 DNMT3A  1788 9606 CDS CCACCAGAAGAAGAGAAGAAT105 TRCN0000021242 NM_004992.2 MECP2  4204 9606 CDSCGTCTGCAAAGAGGAGAAGAT 106 TRCN0000330971 NM_004992.3 MECP2  4204 9606CDS GAGAGCGCAAAGACATTGTTT 107 TRCN0000021241 NM_004992.2 MECP2  42049606 CDS CTGGGAAGTATGATGTGTATT 108 TRCN0000380871 NM_004992.3 MECP2 4204 9606 CDS ACCACCTAAGAAGCCCAAATC 109

What is claimed herein is:
 1. An immune cell produced by a methodcomprising: a) inhibiting Enhancer of Zeste Homolog 1 (EZH1) expressionin the MHPC, wherein MHPC are differentiated from CD34+ hemogenicendothelium cells generated from induced pluripotent stem (iPS) cells;and b) contacting the MHPC with a notch ligand or a stromal cell or bothto promote differentiation into the lymphoid lineage and production of Tlymphocytes or B lymphocytes.
 2. The immune cell of claim 1, wherein theimmune cell is CD4/CD8 double positive or CD8 single positive T cell. 3.The immune cell of claim 2, wherein the immune cell is CD8 singlepositive T cell.
 4. The immune cell of claim 1, wherein Enhancer ofZeste Homolog 1 (EZH1) in the immune cell is inhibited.
 5. The immunecell of claim 1, wherein the immune cell has characteristics ofdefinitive lymphoid cells.
 6. The immune cell of claim 1, wherein theimmune cell comprises an exogenous copy of each of the followingreprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC.
 7. Theimmune cell of claim 1, wherein the immune cell is further geneticallymodified to remove the native T cell receptor (TCR) locus, to deleteclass I or class II major histocompatibility complexes or both, toexpress non-canonical HLA-G or HLA-E or both, or to edit endogenous HLAtherein.
 8. A composition comprising a population of the immune cells ofclaim
 1. 9. The composition of claim 8, further comprising apharmaceutically acceptable carrier.
 10. An immune cell produced by amethod comprising: a) in vitro transfecting myeloid progenitor cellsthat are CD34+CD45+ with an exogenous gene coding copy of each of thefollowing transcription factors: ETS-related gene (ERG), homeobox A9(HOXA9), and retinoic acid receptor (RAR)-related orphan receptor alpha(RORA), wherein the transcription factors are expressed in thetransfected cells to produce a population of multilineage hematopoieticprogenitor cells that have myeloid and erythroid potential and have nolymphoid potential or lymphoid potential that is less than 5%; b)inhibiting Enhancer of Zeste Homolog 1 (EZH1) expression in theresultant population of multilineage hematopoietic progenitor cells toexpand lymphoid potential; and c) differentiating the resultantpopulation of multilineage hematopoietic progenitor cells in thepresence of a notch ligand or supportive stroma or both to promotedifferentiation into the lymphoid lineage and production of Tlymphocytes or B lymphocytes.
 11. The immune cell of claim 10, whereinthe T lymphocyte is CD4/CD8 double positive or CD8 single positive Tcell.
 12. The immune cell of claim 10, wherein the EZH1 in the immunecell is inhibited.
 13. The immune cell of claim 10, wherein the immunecell has characteristics of definitive lymphoid cells.
 14. The immunecell of claim 10, wherein the immune cell comprises an exogenous copy ofeach of the following transcription factors: ERG, HOXA9, and RORA. 15.The immune cell of claim 14, wherein the immune cell further comprisesan exogenous copy of each of the following reprogramming factors: SOX4and MYB.
 16. The immune cell of claim 14, wherein the immune cellfurther comprises an exogenous copy of each of the followingreprogramming factors: NFIA and DACH1.
 17. The immune cell of claim 14,wherein the immune cell further comprises an exogenous copy of each ofthe following reprogramming factors OCT4, SOX2, KLF4 and optionallyc-MYC.
 18. The immune cell of claim 10, wherein the immune cell isfurther genetically modified to remove the native T cell receptor (TCR)locus, to delete class I or class II major histocompatibility complexesor both, to express non-canonical HLA-G or HLA-E or both, or to editendogenous HLA therein.
 19. A composition comprising a population of theimmune cells of claim
 10. 20. A method of treating cancer, autoimmunedisorders, hematological diseases, or other genetic diseases anddisorders, the method comprising administering the immune cell of claim1 to a subject in need thereof.
 21. An ex vivo or in vitro method ofimproving in vivo engraftment of hematopoietic cells in a host, themethod comprising transplanting the immune cell of claim 1 into thehost.